Inhibitors of indoleamine 2,3-dioxygenase and methods of their use

ABSTRACT

There are disclosed compounds that modulate or inhibit the enzymatic activity of indoleamine 2,3-dioxygenase (IDO), pharmaceutical compositions containing said compounds and methods of treating proliferative disorders, such as cancer, viral infections and/or inflammatory disorders utilizing the compounds of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage Application of InternationalPatent Application No. PCT/US2017/031040 filed May 4, 2017, which claimsthe benefit of U.S. Provisional Application No. 62/331,767, filed May 4,2016. Each of these applications is incorporated by reference herein inits entirety.

FIELD OF THE INVENTION

The invention relates generally to compounds that modulate or inhibitthe enzymatic activity of indoleamine 2,3-dioxygenase (IDO),pharmaceutical compositions containing said compounds and methods oftreating proliferative disorders, such as cancer, viral infectionsand/or autoimmune diseases utilizing the compounds of the invention.

BACKGROUND OF THE INVENTION

Indoleamine 2,3-dioxygenase (IDO; also known as IDO1) is an IFN-γ targetgene that plays a role in immunomodulation. IDO is an oxidoreductase andone of two enzymes that catalyze the first and rate-limiting step in theconversion of tryptophan to N-formyl-kynurenine. It exists as a 41 kDmonomer that is found in several cell populations, including immunecells, endothelial cells, and fibroblasts. IDO is relativelywell-conserved between species, with mouse and human sharing 63%sequence identity at the amino acid level. Data derived from its crystalstructure and site-directed mutagenesis show that both substrate bindingand the relationship between the substrate and iron-bound dioxygenaseare necessary for activity. A homolog to IDO (IDO2) has been identifiedthat shares 44% amino acid sequence homology with IDO, but its functionis largely distinct from that of IDO. (See, e.g., Serafini, P. et al.,Semin. Cancer Biol., 16(1):53-65 (February 2006) and Ball, H. J. et al.,Gene, 396(1):203-213 (Jul. 1, 2007)).

IDO plays a major role in immune regulation, and its immunosuppressivefunction manifests in several manners. Importantly, IDO regulatesimmunity at the T cell level, and a nexus exists between IDO andcytokine production. In addition, tumors frequently manipulate immunefunction by upregulation of IDO. Thus, modulation of IDO can have atherapeutic impact on a number of diseases, disorders and conditions.

A pathophysiological link exists between IDO and cancer. Disruption ofimmune homeostasis is intimately involved with tumor growth andprogression, and the production of IDO in the tumor microenvironmentappears to aid in tumor growth and metastasis. Moreover, increasedlevels of IDO activity are associated with a variety of different tumors(Brandacher, G. et al., Clin. Cancer Res., 12(4):1144-1151 (Feb. 15,2006)).

Treatment of cancer commonly entails surgical resection followed bychemotherapy and radiotherapy. The standard treatment regimens showhighly variable degrees of long-term success because of the ability oftumor cells to essentially escape by regenerating primary tumor growthand, often more importantly, seeding distant metastasis. Recent advancesin the treatment of cancer and cancer-related diseases, disorders andconditions comprise the use of combination therapy incorporatingimmunotherapy with more traditional chemotherapy and radiotherapy. Undermost scenarios, immunotherapy is associated with less toxicity thantraditional chemotherapy because it utilizes the patient's own immunesystem to identify and eliminate tumor cells.

In addition to cancer, IDO has been implicated in, among otherconditions, immunosuppression, chronic infections, and autoimmunediseases or disorders (e.g., rheumatoid arthritis). Thus, suppression oftryptophan degradation by inhibition of IDO activity has tremendoustherapeutic value. Moreover, inhibitors of IDO can be used to enhance Tcell activation when the T cells are suppressed by pregnancy,malignancy, or a virus (e.g., HIV). Although their roles are not as welldefined, IDO inhibitors may also find use in the treatment of patientswith neurological or neuropsychiatric diseases or disorders (e.g.,depression).

Small molecule inhibitors of IDO have been developed to treat or preventIDO-related diseases. For example, the IDO inhibitors1-methyl-DL-tryptophan; p-(3-benzofuranyl)-DL-alanine;p-[3-benzo(b)thienyl]-DL-alanine; and 6-nitro-L-tryptophan have beenused to modulate T cell-mediated immunity by altering localextracellular concentrations of tryptophan and tryptophan metabolites(WO 99/29310). Compounds having IDO inhibitory activity are furtherreported in PCT Publication No. WO 2004/094409.

In view of the role played by indoleamine 2,3-dioxygenase in a diversearray of diseases, disorders and conditions, and the limitations (e.g.,efficacy) of current IDO inhibitors, new IDO modulators, andcompositions and methods associated therewith, are needed.

SUMMARY OF THE INVENTION

The invention is directed to compounds of formula I or formula II:

wherein X is CH or N; Q is CH or N; R¹ is H, halo, or C₁-C₆haloalkyl;R^(1A) is H, halo, or C₁-C₆haloalkyl; T is a bond or —O—; Y is CH or N;W is N or —CR⁵—, wherein R⁵ is H or (C₁-C₆alkyl); n is 0, 1, 2, 3, or 4;V is C₁-C₆alkylene optionally substituted with one, two, or three R⁴substituents independently selected from C₁-C₆alkyl and C₃-C₆cycloalkyl;or R⁴ and R⁵, together with the atoms to which they are attached, form aC₃cycloalkyl; R² is H or C₁-C₆alkyl; Z is —NH—, —N(C₁-C₆alkyl), or —O—;R³ is —OH, C₁-C₆alkyl, C₁-C₆haloalkyl, halo, —OC₁-C₆alkyl,—OC₁-C₆haloalkyl, —CN, aryl, or —Oaryl; and R^(3A) is H, —OH,C₁-C₆alkyl, C₁-C₆haloalkyl, halo, —OC₁-C₆alkyl, —OC₁-C₆haloalkyl, —CN,aryl, or —Oaryl;

Also within the scope of the invention are pharmaceutically acceptablesalts, stereoisomers, tautomers, and solvates of the compounds offormula I and formula II.

The invention is also directed to pharmaceutical compositions comprisingone or more compounds of formula I and/or formula II. The invention isalso directed to methods of treating cancer using one or more compoundsof formula I and/or formula II.

DETAILED DESCRIPTION OF THE INVENTION Compounds of the Invention

The present invention is directed to compounds of formula I and formulaII.

According to the disclosure, X is CH or N. In some aspect, X is CH. Inother aspects, X is N.

According to the disclosure Q is CH or N. In some aspects, Q is CH. Inother aspects, Q is N.

In some embodiments of formula I, X is CH and Q is CH. In otherembodiments, X is N and Q is CH. In other embodiments, X is CH and Q isN. In other embodiments, X is N and Q is N.

According to the disclosure, R¹ is H, halo, or C₁-C₆haloalkyl. In otheraspects, R¹ is H, halo, C₁-C₆alkyl, or C₁-C₆haloalkyl. In some aspects,R¹ is H. In some aspects, R¹ is halo (F, Cl, Br, or I), preferably F. Inother aspects, R¹ is C₁-C₆alkyl, for example, methyl, ethyl, isopropyl,butyl, and t-butyl. In yet other aspects, R¹ is C₁-C₆haloalkyl, forexample, —CF₃. In some aspects, R¹ is halo, C₁-C₆alkyl, orC₁-C₆haloalkyl. In other aspects, R¹ is halo or C₁-C₆haloalkyl.

In those aspects of the disclosure comprising compounds of formula I,when R¹ is halo, C₁-C₆alkyl, or C₁-C₆haloalkyl, the R¹ is preferablypresent at the 4-carbon position of the ring. In other aspects of thedisclosure comprising compounds of formula I, when R¹ is halo,C₁-C₆alkyl, or C₁-C₆haloalkyl, the R¹ can be present at the 2-carbonposition of the ring. Preferably, in those aspects of the disclosurecomprising compounds of formula I, when R¹ is F or CF₃ at the 4-carbonposition of the ring.

In those aspects of the disclosure comprising compounds of formula II,when R¹ is halo, C₁-C₆alkyl, or C₁-C₆haloalkyl, the R¹ is preferablypresent at the 2-carbon position of the ring. In other aspects of thedisclosure comprising compounds of formula II, when R¹ is halo,C₁-C₆alkyl, or C₁-C₆haloalkyl, the R¹ can be present at the 3-carbonposition of the ring. Preferably, in those aspects of the disclosurecomprising compounds of formula II, when R¹ is F or CF₃ at the 2-carbonposition of the ring.

According to the disclosure, R^(1A) is H, halo, C₁-C₆alkyl, orC₁-C₆haloalkyl. In preferred aspects, R^(1A) is H. In some aspects,R^(1A) is halo (F, Cl, Br, or I). In other preferred aspects, R^(1A) isC₁-C₆alkyl, for example, methyl, ethyl, isopropyl, butyl, and t-butyl.In yet other aspects, R^(1A) is C₁-C₆haloalkyl, for example, —CF₃.

According to the disclosure, T is a bond or —O—. In preferred aspects, Tis a bond (i.e. T is absent). In other aspects, T- is —O—. In morepreferred aspects, T is a bond when Y is CH or N. In other preferredaspects, T is —O— when Y is CH.

According to the disclosure, Y is CH or N. In some aspects, Y is CH. Inother aspect, Y is N. In other embodiments, Y is —C(C₁-C₆alkyl), forexample, —C(CH₃) or —C(CH₂CH₃).

According to the disclosure, W is N or —CR⁵—, wherein R⁵ is H or(C₁-C₆alkyl). In some aspects, W is —CR⁵— wherein R⁵ is H, i.e., W isCH. In other aspects, W is —CR⁵— wherein R⁵ is (C₁-C₆alkyl), i.e., W is—C(C₁-C₆alkyl)-, for example, —C(CH₃)—, —C(CH₂CH₃)—, —C(i-propyl), or—C(butyl)-. In other aspects, W is N. In some aspects, W is CH or—C(C₁-C₆alkyl)-. In other aspects, W is CH or N. In yet other aspects, Wis N or —C(C₁-C₆alkyl)-.

In some aspects, Y is CH and W is CH. In other aspects, Y is CH and W isN. In other aspects, Y is N and W is CH. In still other aspects, Y is Nand W is N. In some aspects, Y is CH and W is C(C₁-C₆alkyl). In otheraspects, Y is N and W is C(C₁-C₆alkyl).

According to the disclosure, n is 0, 1, 2, 3, or 4. In preferredaspects, n is 2. In other aspects, n is 0. In other aspects, n is 1. Inyet other aspects, n is 3. In still other aspects, n is 4. In someaspects, n is 0 to 2 or 1 to 2. In yet other aspects, n is 1 to 3.

According to the disclosure, V is unsubstituted C₁-C₆alkylene, forexample, unsubstituted C₁-C₅alkylene, C₁-C₄alkylene, C₁-C₃alkylene,C₁-C₂alkylene, or C₁alkylene. In other aspects of the disclosure, V isC₁-C₆alkylene, for example, C₁-C₅alkylene, C₁-C₄alkylene, C₁-C₃alkylene,C₁-C₂alkylene, or C₁alkylene, substituted with one, two, or threesubstituents, preferably 1 or 2 substituents. The substituents can beone or more R⁴. The R⁴ moieties can be independently selected from, forexample, C₁-C₆alkyl (e.g., methyl, ethyl, isopropyl) and C₃-C₆cycloalkyl(e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl). In a preferredaspect, V is a C₁alkylene substituted with one or two, preferably one,C₁-C₆alkyl (e.g., methyl or ethyl) substituents.

In some aspects, W is CR⁵ and V is substituted with one R⁴. In some ofthese aspects, R⁴ and R⁵, together with the atoms to which they areattached, form a C₃cycloalkyl moiety, i.e., a cyclopropyl ring.

According to the disclosure, R² is H or C₁-C₆alkyl. In some aspects, R²is H. In other aspects, R² is C₁-C₆alkyl, for example, methyl, ethyl,isopropyl, butyl, or t-butyl.

According to the disclosure, Z is —NH—, —N(C₁-C₆alkyl)- or —O—. In someaspects, Z is —NH—. In other aspects, Z is —N(C₁-C₆alkyl)-, for example,—N(CH₃)—, —N(CH₂CH₃)—, —N(i-propyl)-, and —N(t-butyl)-. In yet otheraspects, Z is —O—. In some aspects, Z is —NH— or —O—.

According to the disclosure, R³ is H, —OH, C₁-C₆alkyl, C₁-C₆haloalkyl,halo, —OC₁-C₆alkyl, —OC₁-C₆haloalkyl, —CN, aryl, or —Oaryl. In someaspects, R³ is —OH, C₁-C₆alkyl, C₁-C₆haloalkyl, halo, —OC₁-C₆alkyl,—OC₁-C₆haloalkyl, —CN, aryl, or —Oaryl. In some aspects, R³ is H. Insome aspects, R³ is —OH. In some embodiments, R³ is C₁-C₆alkyl, forexample, methyl, ethyl, isopropyl, butyl, or t-butyl. In other aspects,R³ is C₁-C₆haloalkyl, for example, —CF₃. In some aspects, R³ is halo,for example, F, Cl, or Br, with F and Cl being preferred. In someaspects, R³ is —OC₁-C₆alkyl, for example, methoxy, ethoxy, isopropoxy,or butoxy. In other aspects, R³ is —OC₁-C₆haloalkyl, for example, —OCF₃.In some aspects, R³ is —CN. In other embodiments, R³ is aryl, forexample, phenyl or naphthyl. In some aspects, R³ is —Oaryl, for example,—Ophenyl.

According to the disclosure, R^(3A) is H, —OH, C₁-C₆alkyl,C₁-C₆haloalkyl, halo, —OC₁-C₆alkyl, —OC₁-C₆haloalkyl, —CN, aryl, or—Oaryl. In some aspects, R^(3A) is H. In some aspects, R^(3A) is —OH. Insome embodiments, R^(3A) is C₁-C₆alkyl, for example, methyl, ethyl,isopropyl, butyl, or t-butyl. In other aspects, R^(3A) isC₁-C₆haloalkyl, for example, —CF₃. In some aspects, R^(3A) is halo, forexample, F, Cl, or Br, with F and Cl being preferred. In some aspects,R^(3A) is —OC₁-C₆alkyl, for example, methoxy, ethoxy, isopropoxy, orbutoxy. In other aspects, R^(3A) is —OC₁-C₆haloalkyl, for example,—OCF₃. In some aspects, R^(3A) is —CN. In other embodiments, R^(3A) isaryl, for example, phenyl or naphthyl. In some aspects, R^(3A) is—Oaryl, for example, —Ophenyl.

The invention also encompasses the pharmaceutically acceptable salts,stereoisomers, tautomers, and solvates of formulas I and II.

Sub-formulas of formula I and formula II include formulas wherein Y isCH, T is a bond, W is CH, and n is 2 and for example,

wherein X is CH, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is NH. Otherpreferred embodiments of compounds of formulas I-A and II-A are thosewherein X is CH, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is —O—.Other preferred embodiments of compounds of formulas I-A and II-A arethose wherein X is N, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is NH.Other preferred embodiments of compounds of formulas I-A and II-A arethose wherein X is N, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is —O—.In other aspects, X is CH and Q is CH. In some aspects, X is N and Q isCH. In other aspects, X is CH and Q is N. In other aspects, X is N and Qis N. The invention also encompasses the pharmaceutically acceptablesalts, stereoisomers, tautomers, and solvates of formulas I-A and II-A.

Sub-formulas of formula I and formula II include formulas wherein n is2, Y is N, T is a bond, and W is CH, for example,

wherein X is CH, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is NH. Otherpreferred embodiments of compounds of formulas I-B and II-B are thosewherein Xis CH, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is —O—. Otherpreferred embodiments of compounds of formulas I-B and II-B are thosewherein X is N, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is NH. Otherpreferred embodiments of compounds of formulas I-B and II-B are thosewherein X is N, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is —O. Inother aspects, X is CH and Q is CH. In some aspects, X is N and Q is CH.In other aspects, X is CH and Q is N. In other aspects, X is N and Q isN. The invention also encompasses the pharmaceutically acceptable salts,stereoisomers, tautomers, and solvates of formulas I-B and II-B.

Sub-formulas of formula I and formula II include formulas wherein n is2, Y is CH, T is —O—, and W is CH, for example,

wherein X is CH, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is NH. Otherpreferred embodiments of compounds of formulas I-C and II-C are thosewherein Xis CH, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is —O—. Otherpreferred embodiments of compounds of formulas I-B and II-B are thosewherein X is N, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is NH. Otherpreferred embodiments of compounds of formulas I-B and II-B are thosewherein X is N, R⁴ is C₁-C₆alkyl, preferably methyl, and Z is —O—. Inother aspects, X is CH and Q is CH. In some aspects, X is N and Q is CH.In other aspects, X is CH and Q is N. In other aspects, X is N and Q isN. The invention also encompasses the pharmaceutically acceptable salts,stereoisomers, tautomers, and solvates of formulas I-C and II-C.

Other embodiments of the disclosure are directed to compounds of formula1 and formula 2:

wherein

-   -   X is CH or N;    -   Q is CH or N;    -   R¹ is H, halo, or C₁-C₆haloalkyl;    -   R^(1A) is H, halo, or C₁-C₆haloalkyl;    -   T is a bond or —O—;    -   Y is CH or N;    -   W is N or —CR⁵—, wherein R⁵ is H or (C₁-C₆alkyl);    -   n is 0, 1, 2, 3, or 4;    -   V is C₁-C₆alkylene optionally substituted with one, two, or        three R⁴ substituents independently selected from C₁-C₆alkyl and        C₃-C₆cycloalkyl; or R⁴ and R⁵, together with the atoms to which        they are attached, form a C₃cycloalkyl;    -   R² is H or C₁-C₆alkyl;    -   Z is —NH—, —N(C₁-C₆alkyl), or —O—; and    -   B is C₃-C₁₂cycloalkyl optionally substituted with 1, 2, or 3 R        substituents independently selected from —OH, C₁-C₆alkyl,        C₁-C₆haloalkyl, halo, —OC₁-C₆alkyl, —OC₁-C₆haloalkyl, —CN, aryl,        or —Oaryl;        or a pharmaceutically acceptable salt thereof, a stereoisomer        thereof, a tautomer thereof, or a solvate thereof.

In compounds of formula (1) and (2), X is CH or N. In some aspect, X isCH. In other aspects, X is N.

In compounds of formula (1) and (2), Q is CH or N. In some aspects, Q isCH. In other aspects, Q is N.

In compounds of formula (1), Xis CH and Q is CH. In other embodiments,Xis N and Q is CH. In other embodiments, X is CH and Q is N. In otherembodiments, X is N and Q is N.

In compounds of formula (1) and (2), R¹ is H, halo, or C₁-C₆haloalkyl.In other aspects, R¹ is H, halo, C₁-C₆alkyl, or C₁-C₆haloalkyl. In someaspects, R¹ is H. In some aspects, R¹ is halo (F, Cl, Br, or I),preferably F. In other aspects, R¹ is C₁-C₆alkyl, for example, methyl,ethyl, isopropyl, butyl, and t-butyl. In yet other aspects, R¹ isC₁-C₆haloalkyl, for example, —CF₃. In some aspects, R¹ is halo,C₁-C₆alkyl, or C₁-C₆haloalkyl. In other aspects, R¹ is halo orC₁-C₆haloalkyl.

In those aspects of the disclosure comprising compounds of formula (1),when R¹ is halo, C₁-C₆alkyl, or C₁-C₆haloalkyl, the R¹ is preferablypresent at the 4-carbon position of the ring. In other aspects of thedisclosure comprising compounds of formula I, when R¹ is halo,C₁-C₆alkyl, or C₁-C₆haloalkyl, the R¹ can be present at the 2-carbonposition of the ring. Preferably, in those aspects of the disclosurecomprising compounds of formula I, when R¹ is F or CF₃ at the 4-carbonposition of the ring.

In those aspects of the disclosure comprising compounds of formula (2),when R¹ is halo, C₁-C₆alkyl, or C₁-C₆haloalkyl, the R¹ is preferablypresent at the 2-carbon position of the ring. In other aspects of thedisclosure comprising compounds of formula II, when R¹ is halo,C₁-C₆alkyl, or C₁-C₆haloalkyl, the R¹ can be present at the 3-carbonposition of the ring. Preferably, in those aspects of the disclosurecomprising compounds of formula II, when R¹ is F or CF₃ at the 2-carbonposition of the ring.

In compounds of formula (1) and (2), R^(1A) is H, halo, C₁-C₆alkyl, orC₁-C₆haloalkyl. In preferred aspects, R^(1A) is H. In some aspects,R^(1A) is halo (F, Cl, Br, or I). In other preferred aspects, R^(1A) isC₁-C₆alkyl, for example, methyl, ethyl, isopropyl, butyl, and t-butyl.In yet other aspects, R^(1A) is C₁-C₆haloalkyl, for example, —CF₃.

In compounds of formula (1) and (2), T is a bond or —O—. In preferredaspects, T is a bond (i.e. T is absent). In other aspects, T- is —O—. Inmore preferred aspects, T is a bond when Y is CH or N. In otherpreferred aspects, T is —O— when Y is CH.

In compounds of formula (1) and (2), Y is CH or N. In some aspects, Y isCH. In other aspect, Y is N. In other embodiments, Y is —C(C₁-C₆alkyl),for example, —C(CH₃) or —C(CH₂CH₃).

In compounds of formula (1) and (2), W is N or —CR⁵—, wherein R⁵ is H or(C₁-C₆alkyl). In some aspects, W is —CR⁵— wherein R⁵ is H, i.e., W isCH. In other aspects, W is —CR⁵— wherein R⁵ is (C₁-C₆alkyl), i.e., W is—C(C₁-C₆alkyl)-, for example, —C(CH₃)—, —C(CH₂CH₃)—, —O-propyl), or—C(butyl)-. In other aspects, W is N. In some aspects, W is CH or—C(C₁-C₆alkyl)-. In other aspects, W is CH or N. In yet other aspects, Wis N or —C(C₁-C₆alkyl)-.

In compounds of formula (1) and (2), Y is CH and W is CH. In otheraspects, Y is CH and W is N. In other aspects, Y is N and W is CH. Instill other aspects, Y is N and W is N. In some aspects, Y is CH and Wis C(C₁-C₆alkyl). In other aspects, Y is N and W is C(C₁-C₆alkyl).

In compounds of formula (1) and (2), n is 0, 1, 2, 3, or 4. In preferredaspects, n is 2. In other aspects, n is O. In other aspects, n is 1. Inyet other aspects, n is 3. In still other aspects, n is 4. In someaspects, n is 0 to 2 or 1 to 2. In yet other aspects, n is 1 to 3.

In compounds of formula (1) and (2), V is unsubstituted C₁-C₆alkylene,for example, unsubstituted C₁-C₅alkylene, C₁-C₄alkylene, C₁-C₃alkylene,C₁-C₂alkylene, or C₁alkylene. In other aspects of the disclosure, V isC₁-C₆alkylene, for example, C₁-C₅alkylene, C₁-C₄alkylene, C₁-C₃alkylene,C₁-C₂alkylene, or C₁alkylene, substituted with one, two, or threesubstituents, preferably 1 or 2 substituents. The substituents can beone or more R⁴. The R⁴ moieties can be independently selected from, forexample, C₁-C₆alkyl (e.g., methyl, ethyl, isopropyl) and C₃-C₆cycloalkyl(e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl). In a preferredaspect, V is a C₁alkylene substituted with one or two, preferably one,C₁-C₆alkyl (e.g., methyl or ethyl) substituents.

In compounds of formula (1) and (2), W is CR⁵ and V is substituted withone R⁴. In some of these aspects, R⁴ and R⁵, together with the atoms towhich they are attached, form a C₃cycloalkyl moiety, i.e., a cyclopropylring.

In compounds of formula (1) and (2), R² is H or C₁-C₆alkyl. In someaspects, R² is H. In other aspects, R² is C₁-C₆alkyl, for example,methyl, ethyl, isopropyl, butyl, or t-butyl.

According to the disclosure, Z is —NH—, —N(C₁-C₆alkyl)- or —O—. In someaspects, Z is —NH—. In other aspects, Z is —N(C₁-C₆alkyl)-, for example,—N(CH₃)—, —N(CH₂CH₃)—, —N(i-propyl)-, and —N(t-butyl)-. In yet otheraspects, Z is —O—. In some aspects, Z is —NH— or —O—.

In compounds of formula (1) and (2), B is C₃-C₁₂cycloalkyl optionallysubstituted with 1, 2, or 3 R substituents independently selected from—OH, C₁-C₆alkyl, C₁-C₆haloalkyl, halo, —OC₁-C₆alkyl, —OC₁-C₆haloalkyl,—CN, aryl, or —Oaryl. According to some aspects, B is unsubstitutedC₃-C₁₂cycloalkyl, for example, C₃cycloalkyl (e.g., cyclopropyl),C₄cycloalkyl (e.g., cyclobutyl), C₅cycloalkyl (e.g., cyclopentyl),C₆cycloalkyl (e.g., cyclohexyl), C₇cycloalkyl (e.g.,bicyclo[2.2.1]heptanyl), C₈cycloalkyl (bicyclo[2.2.2]octanyl),C₉cycloalkyl, C₁₀cycloalkyl (e.g., adamantyl), C₁₁cycloalkyl, orC₁₂cycloalkyl. In some aspects, B is unsubstituted C₃-C₆cycloalkyl. Inother aspects, B is unsubstituted C₇-C₁₂cycloalkyl or C₇-C₁₀cycloalkyl.In other aspects, B is C₃-C₁₂cycloalkyl substituted with 1, 2, or 3substituents, preferably 1 or 2 substituents (“R” substituents). Forexample, in some aspects, B is substituted C₃cycloalkyl, C₄cycloalkyl,C₅cycloalkyl, C₆cycloalkyl, C₇cycloalkyl, C₈cycloalkyl, C₉cycloalkyl,C₁₀cycloalkyl, C₁₁cycloalkyl, or C₁₂cycloalkyl. In other aspects, B issubstituted C₃-C₆cycloalkyl. In other aspects, B is substitutedC₇-C₁₂cycloalkyl or C₇-C₁₀cycloalkyl. The B substituents, which can bereferred to as one or more R groups, are preferably independentlyselected from —OH, C₁-C₆alkyl, C₁-C₆haloalkyl, halo, —OC₁-C₆alkyl,—OC₁-C₆haloalkyl, —CN, aryl, or —Oaryl. Preferred C₃-C₁₂cycloalkylmoieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, and adamantyl. In someaspects, the C₃-C₁₂cycloalkyl is substituted with at least onesubstituent that is —OH. In some aspects, the C₃-C₁₂cycloalkyl issubstituted with at least one substituent that is C₁-C₆alkyl, forexample, methyl, ethyl, propyl, isopropyl, butyl, s-butyl, t-butyl,pentyl, or hexyl. In other aspects, the C₃-C₁₂cycloalkyl is substitutedwith at least one substituent that is C₁-C₆haloalkyl, for example, —CF₃.In other embodiments, the C₃-C₁₂cycloalkyl is substituted with at leastone substituent that is halo, for example, F, Cl, or Br, with F and Clsubstituents being particularly preferred C₃-C₁₂cycloalkyl substituents.In some aspects, the C₃-C₁₂cycloalkyl is substituted with at least onesubstituent that is —OC₁-C₆alkyl, for example, methoxy, ethoxy, propoxy,isoproxy, butoxy, or t-butoxy. In yet other aspects, theC₃-C₁₂cycloalkyl is substituted with at least one substituent that is—OC₁-C₆haloalkyl, for example, —OCF₃. In some embodiments, theC₃-C₁₂cycloalkyl is substituted with at least one substituent that is—CN. In other aspects, the C₃-C₁₂cycloalkyl is substituted with at leastone substituent that is an aryl moiety, for example, a phenyl ornaphthyl moiety. In some embodiments, the C₃-C₁₂cycloalkyl issubstituted with at least one substituent that is —Oaryl, for example,—Ophenyl.

The invention also encompasses the pharmaceutically acceptable salts,stereoisomers, tautomers, and solvates of formulas (I) and (II), as wellas pharmaceutically acceptable salts, stereoisomers, tautomers, andsolvates of formulas (1) and (2).

In another embodiment, the compounds of the invention have human IDOIC₅₀ values>50 nM. In another embodiment, the compounds of the inventionhave human IDO IC₅₀ values≤50 nM. In another embodiment, the compoundsof the invention have human IDO IC₅₀ values<5 nM.

Other Embodiments of the Invention

In another embodiment, the present invention provides a compositioncomprising one or more compounds of the present invention and/or apharmaceutically acceptable salt thereof, a stereoisomer thereof, atautomer thereof, or a solvate thereof.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atleast one of the compounds of the present invention and/or apharmaceutically acceptable salt thereof, a stereoisomer thereof, atautomer thereof, or a solvate thereof.

In another embodiment, the present invention provides a pharmaceuticalcomposition, comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of at least one of the compounds of thepresent invention and/or a pharmaceutically acceptable salt thereof, astereoisomer thereof, a tautomer thereof, or a solvate thereof.

In another embodiment, the present invention provides a process formaking a compound of the present invention and/or a pharmaceuticallyacceptable salt thereof, a stereoisomer thereof, a tautomer thereof, ora solvate thereof.

In another embodiment, the present invention provides an intermediatefor making a compound of the present invention and/or a pharmaceuticallyacceptable salt thereof, a stereoisomer thereof, a tautomer thereof, ora solvate thereof.

In another embodiment, the present invention provides a method for thetreatment and/or prophylaxis of various types of cancer, viralinfections and/or autoimmune diseases, comprising administering to apatient in need of such treatment and/or prophylaxis a therapeuticallyeffective amount of one or more compounds of the present inventionand/or a pharmaceutically acceptable salt thereof, a stereoisomerthereof or a tautomer thereof, alone, or, optionally, in combinationwith another compound of the present invention and/or at least one othertype of therapeutic agent, such as a chemotherapeutic agent or a signaltransductor inhibitor.

In another embodiment, the present invention provides a compound of thepresent invention, and/or a pharmaceutically acceptable salt thereof, astereoisomer thereof or a tautomer thereof, for use in therapy.

In another embodiment, the present invention provides a combinedpreparation of a compound of the present invention, and/or apharmaceutically acceptable salt thereof, a stereoisomer thereof or atautomer thereof, and additional therapeutic agent(s) for simultaneous,separate or sequential use in therapy.

In another embodiment, the present invention provides a combinedpreparation of a compound of the present invention, and/or apharmaceutically acceptable salt thereof, a stereoisomer thereof or atautomer thereof, and additional therapeutic agent(s) for simultaneous,separate or sequential use in the treatment and/or prophylaxis ofmultiple diseases or disorders associated with the enzymatic activity ofIDO.

In another aspect, the invention provides a method of treating a patientsuffering from or susceptible to a medical condition that is sensitiveto enzymatic activity of IDO. A number of medical conditions can betreated. The method comprises administering to the patient atherapeutically effective amount of a composition comprising a compounddescribed herein and/or a pharmaceutically acceptable salt thereof, astereoisomer thereof or a tautomer thereof. For example, the compoundsdescribed herein may be used to treat or prevent viral infections,proliferative diseases (e.g., cancer), and autoimmune diseases.

Therapeutic Applications

The compounds and pharmaceutical compositions of the present inventionare useful in treating or preventing any disease or conditions that aresensitive to enzymatic activity of IDO. These include viral and otherinfections (e.g., skin infections, GI infection, urinary tractinfections, genito-urinary infections, systemic infections),proliferative diseases (e.g., cancer), and autoimmune diseases (e.g.,rheumatoid arthritis, lupus). The compounds and pharmaceuticalcompositions may be administered to animals, preferably mammals (e.g.,domesticated animals, cats, dogs, mice, rats), and more preferablyhumans. Any method of administration may be used to deliver the compoundor pharmaceutical composition to the patient. In certain embodiments,the compound or pharmaceutical composition is administered orally. Inother embodiments, the compound or pharmaceutical composition isadministered parenterally.

Compounds of the invention can modulate activity of the enzymeindoleamine-2,3-dioxygenase (IDO). The term “modulate” is meant to referto an ability to increase or decrease activity of an enzyme or receptor.Accordingly, compounds of the invention can be used in methods ofmodulating IDO by contacting the enzyme with any one or more of thecompounds or compositions described herein. In some embodiments,compounds of the present invention can act as inhibitors of IDO. Infurther embodiments, the compounds of the invention can be used tomodulate activity of IDO in cell or in an individual in need ofmodulation of the enzyme by administering a modulating (e.g.,inhibiting) amount of a compound of the invention.

Compounds of the invention can inhibit activity of the enzymeindoleamine-2,3-dioxygenase (IDO). For example, the compounds of theinvention can be used to inhibit activity of IDO in cell or in anindividual in need of modulation of the enzyme by administering aninhibiting amount of a compound of the invention.

The present invention further provides methods of inhibiting thedegradation of tryptophan in a system containing cells expressing IDOsuch as a tissue, living organism, or cell culture. In some embodiments,the present invention provides methods of altering (e.g., increasing)extracellular tryptophan levels in a mammal by administering aneffective amount of a compound of composition provided herein. Methodsof measuring tryptophan levels and tryptophan degradation are routine inthe art.

The present invention further provides methods of inhibitingimmunosuppression such as IDO-mediated immunosuppression in a patient byadministering to the patient an effective amount of a compound orcomposition recited herein. IDO-mediated immunosuppression has beenassociated with, for example, cancers, tumor growth, metastasis, viralinfection, and viral replication.

The present invention further provides methods of treating diseasesassociated with activity or expression, including abnormal activityand/or overexpression, of IDO in an individual (e.g., patient) byadministering to the individual in need of such treatment atherapeutically effective amount or dose of a compound of the presentinvention or a pharmaceutical composition thereof. Example diseases caninclude any disease, disorder or condition that is directly orindirectly linked to expression or activity of the IDO enzyme, such asover expression or abnormal activity. An IDO-associated disease can alsoinclude any disease, disorder or condition that can be prevented,ameliorated, or cured by modulating enzyme activity. Examples ofIDO-associated diseases include cancer, viral infection such as HIVinfection, HCV infection, depression, neurodegenerative disorders suchas Alzheimer's disease and Huntington's disease, trauma, age-relatedcataracts, organ transplantation (e.g., organ transplant rejection), andautoimmune diseases including asthma, rheumatoid arthritis, multiplesclerosis, allergic inflammation, inflammatory bowel disease, psoriasisand systemic lupus erythematosus.

As used herein, the term “cell” is meant to refer to a cell that is invitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can bepart of a tissue sample excised from an organism such as a mammal. Insome embodiments, an in vitro cell can be a cell in a cell culture. Insome embodiments, an in vivo cell is a cell living in an organism suchas a mammal.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” the IDO enzyme with a compound of the inventionincludes the administration of a compound of the present invention to anindividual or patient, such as a human, having IDO, as well as, forexample, introducing a compound of the invention into a samplecontaining a cellular or purified preparation containing the IDO enzyme.

The term “IDO inhibitor” refers to an agent capable of inhibiting theactivity of indoleamine 2,3-dioxygenase (IDO) and thereby reversingIDO-mediated immunosuppression. The IDO inhibitor may inhibit IDO1and/or IDO2 (INDOL1). An IDO inhibitor may be a reversible orirreversible IDO inhibitor. “A reversible IDO inhibitor” is a compoundthat reversibly inhibits IDO enzyme activity either at the catalyticsite or at a non-catalytic site and “an irreversible IDO inhibitor” is acompound that irreversibly destroys IDO enzyme activity.

Types of cancers that may be treated with the compounds of thisinvention include, but are not limited to, brain cancers, skin cancers,bladder cancers, ovarian cancers, breast cancers, gastric cancers,pancreatic cancers, prostate cancers, colon cancers, blood cancers, lungcancers and bone cancers. Examples of such cancer types includeneuroblastoma, intestine carcinoma such as rectum carcinoma, coloncarcinoma, familiar adenomatous polyposis carcinoma and hereditarynon-polyposis colorectal cancer, esophageal carcinoma, labial carcinoma,larynx carcinoma, hypopharynx carcinoma, tongue carcinoma, salivarygland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, renal carcinoma, kidneyparenchymal carcinoma, ovarian carcinoma, cervix carcinoma, uterinecorpus carcinoma, endometrium carcinoma, chorion carcinoma, pancreaticcarcinoma, prostate carcinoma, testis carcinoma, breast carcinoma,urinary carcinoma, melanoma, brain tumors such as glioblastoma,astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermaltumors, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acutelymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acutemyeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cellleukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellularcarcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lungcarcinoma, non-small cell lung carcinoma, multiple myeloma, basalioma,teratoma, retinoblastoma, choroid melanoma, seminoma, rhabdomyosarcoma,craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma,liposarcoma, fibrosarcoma, Ewing sarcoma and plasmacytoma.

Thus, according to another embodiment, the invention provides a methodof treating an autoimmune disease by providing to a patient in needthereof a compound or composition of the present invention. Examples ofsuch autoimmune diseases include, but are not limited to, collagendiseases such as rheumatoid arthritis, systemic lupus erythematosus,Sharp's syndrome, CREST syndrome (calcinosis, Raynaud's syndrome,esophageal dysmotility, telangiectasia), dermatomyositis, vasculitis(Morbus Wegener's) and Sjogren's syndrome, renal diseases such asGoodpasture's syndrome, rapidly-progressing glomerulonephritis andmembranoproliferative glomerulonephritis type II, endocrine diseasessuch as type-I diabetes, autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), autoimmuneparathyroidism, pernicious anemia, gonad insufficiency, idiopathicMorbus Addison's, hyperthyreosis, Hashimoto's thyroiditis and primarymyxedema, skin diseases such as pemphigus vulgaris, bullous pemphigoid,herpes gestationis, epidermolysis bullosa and erythema multiforme major,liver diseases such as primary biliary cirrhosis, autoimmunecholangitis, autoimmune hepatitis type-1, autoimmune hepatitis type-2,primary sclerosing cholangitis, neuronal diseases such as multiplesclerosis, myasthenia gravis, myasthenic Lambert-Eaton syndrome,acquired neuromyotomy, Guillain-Barré syndrome (Muller-Fischersyndrome), stiff-man syndrome, cerebellar degeneration, ataxia,opsoclonus, sensoric neuropathy and achalasia, blood diseases such asautoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (MorbusWerlhof), infectious diseases with associated autoimmune reactions suchas AIDS, malaria and Chagas disease.

One or more additional pharmaceutical agents or treatment methods suchas, for example, anti-viral agents, chemotherapeutics or otheranticancer agents, immune enhancers, immunosuppressants, radiation,anti-tumor and anti-viral vaccines, cytokine therapy (e.g., IL2 andGM-CSF), and/or tyrosine kinase inhibitors can be optionally used incombination with the compounds of the present invention for treatment ofIDO-associated diseases, disorders or conditions. The agents can becombined with the present compounds in a single dosage form, or theagents can be administered simultaneously or sequentially as separatedosage forms.

Suitable chemotherapeutic or other anticancer agents include, forexample, alkylating agents (including, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes) such as uracil mustard, chlormethine, cyclophosphamide(CYTOXAN®), ifosfamide, melphalan, chlorambucil, pipobroman,triethylene-melamine, triethylenethiophosphoramine, busulfan,carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

In the treatment of melanoma, suitable agents for use in combinationwith the compounds of the present invention include: dacarbazine (DTIC),optionally, along with other chemotherapy drugs such as carmustine(BCNU) and cisplatin; the “Dartmouth regimen”, which consists of DTIC,BCNU, cisplatin and tamoxifen; a combination of cisplatin, vinblastine,and DTIC, temozolomide or YERVOY®. Compounds according to the inventionmay also be combined with immunotherapy drugs, including cytokines suchas interferon alpha, interleukin 2, and tumor necrosis factor (TNF) inthe treatment of melanoma.

Compounds of the invention may also be used in combination with vaccinetherapy in the treatment of melanoma. Anti-melanoma vaccines are, insome ways, similar to the anti-virus vaccines which are used to preventdiseases caused by viruses such as polio, measles, and mumps. Weakenedmelanoma cells or parts of melanoma cells called antigens may beinjected into a patient to stimulate the body's immune system to destroymelanoma cells.

Melanomas that are confined to the arms or legs may also be treated witha combination of agents including one or more compounds of theinvention, using a hyperthermic isolated limb perfusion technique. Thistreatment protocol temporarily separates the circulation of the involvedlimb from the rest of the body and injects high doses of chemotherapyinto the artery feeding the limb, thus providing high doses to the areaof the tumor without exposing internal organs to these doses that mightotherwise cause severe side effects. Usually the fluid is warmed to 102°to 104° F. Melphalan is the drug most often used in this chemotherapyprocedure. This can be given with another agent called tumor necrosisfactor (TNF).

Suitable chemotherapeutic or other anticancer agents include, forexample, antimetabolites (including, without limitation, folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors) such as methotrexate, 5-fluorouracil, floxuridine,cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate,pentostatine, and gemcitabine.

Suitable chemotherapeutic or other anticancer agents further include,for example, certain natural products and their derivatives (forexample, vinca alkaloids, antitumor antibiotics, enzymes, lymphokinesand epipodophyllotoxins) such as vinblastine, vincristine, vindesine,bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin,idarubicin, ara-C, paclitaxel (Taxol), mithramycin, deoxycoformycin,mitomycin-C, L-asparaginase, interferons (especially IFN-α), etoposide,and teniposide.

Other cytotoxic agents include navelbene, CPT-11, anastrazole,letrazole, capecitabine, reloxafine, and droloxafine.

Also suitable are cytotoxic agents such as epidophyllotoxin; anantineoplastic enzyme; a topoisomerase inhibitor; procarbazine;mitoxantrone; platinum coordination complexes such as cisplatin andcarboplatin; biological response modifiers; growth inhibitors;antihormonal therapeutic agents; leucovorin; tegafur; and haematopoieticgrowth factors.

Other anticancer agent(s) include antibody therapeutics such astrastuzumab (HERCEPTIN®), antibodies to costimulatory molecules such asCTLA-4, 4-1BB and PD-1, or antibodies to cytokines (IL-10 or TGF-β).

Other anticancer agents also include those that block immune cellmigration such as antagonists to chemokine receptors, including CCR2 andCCR4.

Other anticancer agents also include those that augment the immunesystem such as adjuvants or adoptive T cell transfer.

Anticancer vaccines include dendritic cells, synthetic peptides, DNAvaccines and recombinant viruses.

The pharmaceutical composition of the invention may optionally includeat least one signal transduction inhibitor (STI). A “signal transductioninhibitor” is an agent that selectively inhibits one or more vital stepsin signaling pathways, in the normal function of cancer cells, therebyleading to apoptosis. Suitable STIs include, but are not limited to: (i)bcr/abl kinase inhibitors such as, for example, STI 571 (GLEEVEC®); (ii)epidermal growth factor (EGF) receptor inhibitors such as, for example,kinase inhibitors (IRESSA®, SSI-774) and antibodies (Imclone: C225[Goldstein et al., Clin. Cancer Res., 1:1311-1318 (1995)], and Abgenix:ABX-EGF); (iii) her-2/neu receptor inhibitors such as farnesyltransferase inhibitors (FTI) such as, for example, L-744,832 (Kohl etal., Nat. Med., 1(8):792-797 (1995)); (iv) inhibitors of Akt familykinases or the Akt pathway, such as, for example, rapamycin (see, forexample, Sekulic et al., Cancer Res., 60:3504-3513 (2000)); (v) cellcycle kinase inhibitors such as, for example, flavopiridol and UCN-01(see, for example, Sausville, Curr. Med. Chem. Anti-Canc. Agents,3:47-56 (2003)); and (vi) phosphatidyl inositol kinase inhibitors suchas, for example, LY294002 (see, for example, Vlahos et al., J. Biol.Chem., 269:5241-5248 (1994)). Alternatively, at least one STI and atleast one IDO inhibitor may be in separate pharmaceutical compositions.In a specific embodiment of the present invention, at least one IDOinhibitor and at least one STI may be administered to the patientconcurrently or sequentially. In other words, at least one IDO inhibitormay be administered first, at least one STI may be administered first,or at least one IDO inhibitor and at least one STI may be administeredat the same time. Additionally, when more than one IDO inhibitor and/orSTI is used, the compounds may be administered in any order.

The present invention further provides a pharmaceutical composition forthe treatment of a chronic viral infection in a patient comprising atleast one IDO inhibitor, optionally, at least one chemotherapeutic drug,and, optionally, at least one antiviral agent, in a pharmaceuticallyacceptable carrier. The pharmaceutical compositions may include at leastone IDO inhibitor of the instant invention in addition to at least oneestablished (known) IDO inhibitor. In a specific embodiment, at leastone of the IDO inhibitors of the pharmaceutical composition is selectedfrom the group consisting of compounds of formulas I and (II).

Also provided is a method for treating a chronic viral infection in apatient by administering an effective amount of the above pharmaceuticalcomposition.

In a specific embodiment of the present invention, at least one IDOinhibitor and at least one chemotherapeutic agent may be administered tothe patient concurrently or sequentially. In other words, at least oneIDO inhibitor may be administered first, at least one chemotherapeuticagent may be administered first, or at least one IDO inhibitor and theat least one STI may be administered at the same time. Additionally,when more than one IDO inhibitor and/or chemotherapeutic agent is used,the compounds may be administered in any order. Similarly, any antiviralagent or STI may also be administered at any point in comparison to theadministration of an IDO inhibitor.

Chronic viral infections that may be treated using the presentcombinatorial treatment include, but are not limited to, diseases causedby: hepatitis C virus (HCV), human papilloma virus (HPV),cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus(EBV), varicella zoster virus, Coxsackie virus, human immunodeficiencyvirus (HIV). Notably, parasitic infections (e.g., malaria) may also betreated by the above methods wherein compounds known to treat theparasitic conditions are optionally added in place of the antiviralagents.

In yet another embodiment, the pharmaceutical compositions comprising atleast one IDO inhibitor of the instant invention may be administered toa patient to prevent arterial restenosis, such as after balloonendoscopy or stent placement. In a particular embodiment, thepharmaceutical composition further comprises at least one taxane (e.g.,paclitaxel (Taxol); see, e.g., Scheller et al., Circulation, 110:810-814(2004)).

Suitable antiviral agents contemplated for use in combination with thecompounds of the present invention can comprise nucleoside andnucleotide reverse transcriptase inhibitors (NRTIs), non-nucleosidereverse transcriptase inhibitors (NNRTIs), protease inhibitors and otherantiviral drugs.

Examples of suitable NRTIs include zidovudine (AZT); didanosine (ddl);zalcitabine (ddC); stavudine (d4T); lamivudine (3TC); abacavir(1592U89); adefovir dipivoxil [bis(POM)-PMEA]; lobucavir (BMS-180194);BCH-I0652; emtricitabine [(−)-FTC]; beta-L-FD4 (also called beta-L-D4Cand named beta-L-2′,3′-dicleoxy-5-fluoro-cytidene); DAPD,((−)-beta-D-2,6-diamino-purine dioxolane); and lodenosine (FddA).Typical suitable NNRTIs include nevirapine (BI-RG-587); delaviradine(BHAP, U-90152); efavirenz (DMP-266); PNU-142721; AG-1549; MKC-442(1-(ethoxy-methyl)-5-(1-methylethyl)-6-(phenylmethyl)-(2,4(1H,3H)-pyrimidinedione);and (+)-calanolide A (NSC-675451) and B. Typical suitable proteaseinhibitors include saquinavir (Ro 31-8959); ritonavir (ABT-538);indinavir (MK-639); nelfinavir (AG-1343); amprenavir (141W94); lasinavir(BMS-234475); DMP-450; BMS-2322623; ABT-378; and AG-1549. Otherantiviral agents include hydroxyurea, ribavirin, IL-2, IL-12,pentafuside and Yissum Project No. 11607.

Combination with an Immuno-Oncology Agent

Further provided herein are methods of treatment wherein a compound offormula I or formula II is administered with one or more immuno-oncologyagents. The immuno-oncology agents used herein, also known as cancerimmunotherapies, are effective to enhance, stimulate, and/or upregulateimmune responses in a subject.

In one aspect, the Compound of formula I or formula II is sequentiallyadministered prior to administration of the immuno-oncology agent. Inanother aspect, the Compound of formula I or formula II is administeredconcurrently with the immunology-oncology agent. In yet another aspect,the Compound of formula I or formula II is sequentially administeredafter administration of the immuno-oncology agent.

In another aspect, the Compound of formula I or formula II may beco-formulated with an immuno-oncology agent.

Immuno-oncology agents include, for example, a small molecule drug,antibody, or other biologic or small molecule. Examples of biologicimmuno-oncology agents include, but are not limited to, cancer vaccines,antibodies, and cytokines. In one aspect, the antibody is a monoclonalantibody. In another aspect, the monoclonal antibody is humanized orhuman.

In one aspect, the immuno-oncology agent is (i) an agonist of astimulatory (including a co-stimulatory) receptor or (ii) an antagonistof an inhibitory (including a co-inhibitory) signal on T cells, both ofwhich result in amplifying antigen-specific T cell responses (oftenreferred to as immune checkpoint regulators).

Certain of the stimulatory and inhibitory molecules are members of theimmunoglobulin super family (IgSF). One important family ofmembrane-bound ligands that bind to co-stimulatory or co-inhibitoryreceptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1),B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6.Another family of membrane bound ligands that bind to co-stimulatory orco-inhibitory receptors is the TNF family of molecules that bind tocognate TNF receptor family members, which includes CD40 and CD40L,OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB),TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK,RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR,LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1,Lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, Lymphotoxin α 1β2, FAS, FASL,RELT, DR6, TROY, NGFR.

In another aspect, the immuno-oncology agent is a cytokine that inhibitsT cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and otherimmunosuppressive cytokines) or a cytokine that stimulates T cellactivation, for stimulating an immune response.

In one aspect, T cell responses can be stimulated by a combination ofthe Compound of Formula I or formula II and one or more of (i) anantagonist of a protein that inhibits T cell activation (e.g., immunecheckpoint inhibitors) such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3,Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56,VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and (ii) anagonist of a protein that stimulates T cell activation such as B7-1,B7-2, CD28, 4-IBB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR,GITRL, CD70, CD27, CD40, DR3 and CD28H.

Other agents that can be combined with the Compound of formula I orformula II for the treatment of cancer include antagonists of inhibitoryreceptors on NK cells or agonists of activating receptors on NK cells.For example, the Compound of formula I or formula II can be combinedwith antagonists of KIR, such as lirilumab.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155 (WO11/70024, WO 11/107553, WO 11/131407, WO 13/87699, WO 13/119716, WO13/132044) or FPA-008 (WO 11/140249, WO 13/169264, WO 14/036357).

In another aspect, the Compound of formula I or formula II can be usedwith one or more of agonistic agents that ligate positive costimulatoryreceptors, blocking agents that attenuate signaling through inhibitoryreceptors, antagonists, and one or more agents that increasesystemically the frequency of anti-tumor T cells, agents that overcomedistinct immune suppressive pathways within the tumor microenvironment(e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1interactions), deplete or inhibit Tregs (e.g., using an anti-CD25monoclonal antibody (e.g., daclizumab) or by ex vivo anti-CD25 beaddepletion), inhibit metabolic enzymes such as IDO, or reverse/prevent Tcell anergy or exhaustion) and agents that trigger innate immuneactivation and/or inflammation at tumor sites.

In one aspect, the immuno-oncology agent is a CTLA-4 antagonist, such asan antagonistic CTLA-4 antibody. Suitable CTLA-4 antibodies include, forexample, YERVOY® (ipilimumab) or tremelimumab.

In another aspect, the immuno-oncology agent is a PD-1 antagonist, suchas an antagonistic PD-1 antibody. Suitable PD-1 antibodies include, forexample, OPDIVO® (nivolumab), KEYTRUDA® (pembrolizumab), or MEDI-0680(AMP-514; WO 2012/145493). The immuno-oncology agent may also includepidilizumab (CT-011), though its specificity for PD-1 binding has beenquestioned. Another approach to target the PD-1 receptor is therecombinant protein composed of the extracellular domain of PD-L2(B7-DC) fused to the Fc portion of IgG1, called AMP-224

In another aspect, the immuno-oncology agent is a PD-L1 antagonist, suchas an antagonistic PD-L1 antibody. Suitable PD-L1 antibodies include,for example, MPDL3280A (RG7446; WO 2010/077634), durvalumab (MEDI4736),BMS-936559 (WO 2007/005874), and MSB0010718C (WO 2013/79174).

In another aspect, the immuno-oncology agent is a LAG-3 antagonist, suchas an antagonistic LAG-3 antibody. Suitable LAG3 antibodies include, forexample, BMS-986016 (WO 10/19570, WO 14/08218), or IMP-731 or IMP-321(WO 08/132601, WO 09/44273).

In another aspect, the immuno-oncology agent is a CD137 (4-1BB) agonist,such as an agonistic CD137 antibody. Suitable CD137 antibodies include,for example, urelumab and PF-05082566 (WO 12/32433).

In another aspect, the immuno-oncology agent is a GITR agonist, such asan agonistic GITR antibody. Suitable GITR antibodies include, forexample, BMS-986153, BMS-986156, TRX-518 (WO 06/105021, WO 09/009116)and MK-4166 (WO 11/028683).

In another aspect, the immuno-oncology agent is an IDO antagonist.Suitable IDO antagonists include, for example, INCB-024360 (WO2006/122150, WO 07/75598, WO 08/36653, WO 08/36642), indoximod, orNLG-919 (WO 09/73620, WO 09/1156652, WO 11/56652, WO 12/142237).

In another aspect, the immuno-oncology agent is an OX40 agonist, such asan agonistic OX40 antibody. Suitable OX40 antibodies include, forexample, MEDI-6383 or MEDI-6469.

In another aspect, the immuno-oncology agent is an OX40L antagonist,such as an antagonistic OX40 antibody. Suitable OX40L antagonistsinclude, for example, RG-7888 (WO 06/029879).

In another aspect, the immuno-oncology agent is a CD40 agonist, such asan agonistic CD40 antibody. In yet another embodiment, theimmuno-oncology agent is a CD40 antagonist, such as an antagonistic CD40antibody. Suitable CD40 antibodies include, for example, lucatumumab ordacetuzumab.

In another aspect, the immuno-oncology agent is a CD27 agonist, such asan agonistic CD27 antibody. Suitable CD27 antibodies include, forexample, varlilumab.

In another aspect, the immuno-oncology agent is MGA271 (to B7H3) (WO11/109400).

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of IDO-associated diseases ordisorders, obesity, diabetes and other diseases referred to herein whichinclude one or more containers containing a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of theinvention. Such kits can further include, if desired, one or more ofvarious conventional pharmaceutical kit components, such as, forexample, containers with one or more pharmaceutically acceptablecarriers, additional containers, as will be readily apparent to thoseskilled in the art. Instructions, either as inserts or as labels,indicating quantities of the components to be administered, guidelinesfor administration, and/or guidelines for mixing the components, canalso be included in the kit.

The combination therapy is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single dosage form having afixed ratio of each therapeutic agent or in multiple, single dosageforms for each of the therapeutic agents. Sequential or substantiallysimultaneous administration of each therapeutic agent can be effected byany appropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally or all therapeutic agents may be administeredby intravenous injection. Combination therapy also can embrace theadministration of the therapeutic agents as described above in furthercombination with other biologically active ingredients and non-drugtherapies (e.g., surgery or radiation treatment). Where the combinationtherapy further comprises a non-drug treatment, the non-drug treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andnon-drug treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

Pharmaceutical Compositions and Dosing

The invention also provides pharmaceutically acceptable compositionswhich comprise a therapeutically effective amount of one or more of thecompounds of formula I and/or Formula II, formulated together with oneor more pharmaceutically acceptable carriers (additives) and/ordiluents, and optionally, one or more additional therapeutic agentsdescribed above.

The compounds of this invention can be administered for any of the usesdescribed herein by any suitable means, for example, orally, such astablets, capsules (each of which includes sustained release or timedrelease formulations), pills, powders, granules, elixirs, tinctures,suspensions (including nanosuspensions, microsuspensions, spray-drieddispersions), syrups, and emulsions; sublingually; buccally;parenterally, such as by subcutaneous, intravenous, intramuscular, orintrasternal injection, or infusion techniques (e.g., as sterileinjectable aqueous or non-aqueous solutions or suspensions); nasally,including administration to the nasal membranes, such as by inhalationspray; topically, such as in the form of a cream or ointment; orrectally such as in the form of suppositories. They can be administeredalone, but generally will be administered with a pharmaceutical carrierselected on the basis of the chosen route of administration and standardpharmaceutical practice.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient.

The term “pharmaceutical composition” means a composition comprising acompound of the invention in combination with at least one additionalpharmaceutically acceptable carrier. A “pharmaceutically acceptablecarrier” refers to media generally accepted in the art for the deliveryof biologically active agents to animals, in particular, mammals,including, i.e., adjuvant, excipient or vehicle, such as diluents,preserving agents, fillers, flow regulating agents, disintegratingagents, wetting agents, emulsifying agents, suspending agents,sweetening agents, flavoring agents, perfuming agents, antibacterialagents, antifungal agents, lubricating agents and dispensing agents,depending on the nature of the mode of administration and dosage forms.

Pharmaceutically acceptable carriers are formulated according to anumber of factors well within the purview of those of ordinary skill inthe art. These include, without limitation: the type and nature of theactive agent being formulated; the subject to which the agent-containingcomposition is to be administered; the intended route of administrationof the composition; and the therapeutic indication being targeted.Pharmaceutically acceptable carriers include both aqueous andnon-aqueous liquid media, as well as a variety of solid and semi-soliddosage forms. Such carriers can include a number of differentingredients and additives in addition to the active agent, suchadditional ingredients being included in the formulation for a varietyof reasons, e.g., stabilization of the active agent, binders, etc., wellknown to those of ordinary skill in the art. Descriptions of suitablepharmaceutically acceptable carriers, and factors involved in theirselection, are found in a variety of readily available sources such as,for example, Allen, Jr., L.V. et al., Remington: The Science andPractice of Pharmacy (2 Volumes), 22nd Edition, Pharmaceutical Press(2012).

The dosage regimen for the compounds of the present invention will, ofcourse, vary depending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the species, age, sex, health, medical condition, andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; the route ofadministration, the renal and hepatic function of the patient, and theeffect desired.

By way of general guidance, the daily oral dosage of each activeingredient, when used for the indicated effects, will range betweenabout 0.001 to about 5000 mg per day, preferably between about 0.01 toabout 1000 mg per day, and most preferably between about 0.1 to about250 mg per day. Intravenously, the most preferred doses will range fromabout 0.01 to about 10 mg/kg/minute during a constant rate infusion.Compounds of this invention may be administered in a single daily dose,or the total daily dosage may be administered in divided doses of two,three, or four times daily.

The compounds are typically administered in admixture with suitablepharmaceutical diluents, excipients, or carriers (collectively referredto herein as pharmaceutical carriers) suitably selected with respect tothe intended form of administration, e.g., oral tablets, capsules,elixirs, and syrups, and consistent with conventional pharmaceuticalpractices.

Dosage forms (pharmaceutical compositions) suitable for administrationmay contain from about 1 milligram to about 2000 milligrams of activeingredient per dosage unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.1-95% by weight based on the total weight of the composition.

A typical capsule for oral administration contains at least one of thecompounds of the present invention (250 mg), lactose (75 mg), andmagnesium stearate (15 mg). The mixture is passed through a 60 meshsieve and packed into a No. 1 gelatin capsule.

A typical injectable preparation is produced by aseptically placing atleast one of the compounds of the present invention (250 mg) into avial, aseptically freeze-drying and sealing. For use, the contents ofthe vial are mixed with 2 mL of physiological saline, to produce aninjectable preparation.

The present invention includes within its scope pharmaceuticalcompositions comprising, as an active ingredient, a therapeuticallyeffective amount of at least one of the compounds of the presentinvention, alone or in combination with a pharmaceutical carrier.Optionally, compounds of the present invention can be used alone, incombination with other compounds of the invention, or in combinationwith one or more other therapeutic agent(s), e.g., an anticancer agentor other pharmaceutically active material.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain aspects of the invention,dosing is one administration per day.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

Definitions

Unless specifically stated otherwise herein, references made in thesingular may also include the plural. For example, “a” and “an” mayrefer to either one, or one or more.

Unless otherwise indicated, any heteroatom with unsatisfied valences isassumed to have hydrogen atoms sufficient to satisfy the valences.

Throughout the specification and the appended claims, a given chemicalformula or name shall encompass all stereo and optical isomers andracemates thereof where such isomers exist. Unless otherwise indicated,all chiral (enantiomeric and diastereomeric) and racemic forms arewithin the scope of the invention. Many geometric isomers of C═C doublebonds, C═N double bonds, ring systems, and the like can also be presentin the compounds, and all such stable isomers are contemplated in thepresent invention. Cis- and trans- (or E- and Z-) geometric isomers ofthe compounds of the present invention are described and may be isolatedas a mixture of isomers or as separated isomeric forms. The presentcompounds can be isolated in optically active or racemic forms.Optically active forms may be prepared by resolution of racemic forms orby synthesis from optically active starting materials. All processesused to prepare compounds of the present invention and intermediatesmade therein are considered to be part of the present invention. Whenenantiomeric or diastereomeric products are prepared, they may beseparated by conventional methods, for example, by chromatography orfractional crystallization. Depending on the process conditions the endproducts of the present invention are obtained either in free (neutral)or salt form. Both the free form and the salts of these end products arewithin the scope of the invention. If so desired, one form of a compoundmay be converted into another form. A free base or acid may be convertedinto a salt; a salt may be converted into the free compound or anothersalt; a mixture of isomeric compounds of the present invention may beseparated into the individual isomers. Compounds of the presentinvention, free form and salts thereof, may exist in multiple tautomericforms, in which hydrogen atoms are transposed to other parts of themolecules and the chemical bonds between the atoms of the molecules areconsequently rearranged. It should be understood that all tautomericforms, insofar as they may exist, are included within the invention.

For purposes of clarity and in accordance with standard convention inthe art, the symbol

is used in formulas and tables to show the bond that is the point ofattachment of the moiety or substituent to the core/nucleus of thestructure.

Additionally, for purposes of clarity, where a substituent has a dash(—) that is not between two letters or symbols; this is used to indicatea point of attachment for a substituent. For example, —CONH₂ is attachedthrough the carbon atom.

Additionally, for purposes of clarity, when there is no substituentshown at the end of a solid line, this indicates that there is a methyl(CH₃) group connected to the bond.

As used herein, the terms “alkyl” and “alkylene” (also refered to as“alk”) are intended to include both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms. For example, “C₁-C₆ alkyl” or “C₁₋₆ alkyl” denotes alkylhaving 1 to 6 carbon atoms. Example alkyl groups include, but are notlimited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl andisopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), and pentyl (e.g.,n-pentyl, isopentyl, neopentyl). “C₁-C₆alkylene” denotes alkylene having1 to 6 carbon atoms. Example alkylene groups include, but are notlimited to, methylene (—CH₂—), ethylene (—CH₂CH₂—), and the like.

As used herein, “aryl” refers to an aromatic ring system which includes,but not limited to phenyl, biphenyl, indanyl, 1-naphthyl, 2-naphthyl andterahydronaphthyl.

“Halo” or “halogen” includes fluoro, chloro, bromo, and iodo.

“Haloalkyl” is intended to include both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms, substituted with 1 or more halogens. Examples of haloalkylinclude, but are not limited to, fluoromethyl, difluoromethyl,trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl,2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl. Examplesof haloalkyl also include “fluoroalkyl” that is intended to include bothbranched and straight-chain saturated aliphatic hydrocarbon groupshaving the specified number of carbon atoms, substituted with 1 or morefluorine atoms.

The term “cycloalkyl” refers to hydrocarbon rings having the indicatednumber of ring atoms, e.g., C₃-C₆cycloalkyl or C₃-C₁₂cycloalkyl, andbeing fully saturated. “Cycloalkyl” is also meant to refer to bicyclicand polycylic hydrocarbon rings such as, for example,bicyclo[2.2.1]heptane, bicyclo[2.2.2] octane, and adamantyl. C₃₋₆cycloalkyl is intended to include C₃, C₄, C₅, and C₆ cycloalkyl groups.Example cycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and norbornyl.

As referred to herein, the term “substituted” means that at least onehydrogen atom is replaced with a non-hydrogen group, provided thatnormal valencies are maintained and that the substitution results in astable compound.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic groups such as amines; and alkali or organic saltsof acidic groups such as carboxylic acids. The pharmaceuticallyacceptable salts include the conventional non-toxic salts or thequaternary ammonium salts of the parent compound formed, for example,from non-toxic inorganic or organic acids. For example, suchconventional non-toxic salts include those derived from inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, andnitric; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, andisethionic, and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Allen, Jr., L.V.,ed., Remington: The Science and Practice of Pharmacy, 22nd Edition,Pharmaceutical Press, London, UK (2012). The disclosure of which ishereby incorporated by reference.

In addition, compounds of formula I and formula II may have prodrugforms. Any compound that will be converted in vivo to provide thebioactive agent (i.e., a compound of formula I or II) is a prodrugwithin the scope and spirit of the invention. Various forms of prodrugsare well known in the art. For examples of such prodrug derivatives,see:

-   a) Bundgaard, H., ed., Design of Prodrugs, Elsevier (1985), and    Widder, K. et al., eds., Methods in Enzymology, 112:309-396,    Academic Press (1985);-   b) Bundgaard, H., Chapter 5: “Design and Application of Prodrugs”, A    Textbook of Drug Design and Development, pp. 113-191,    Krogsgaard-Larsen, P. et al., eds., Harwood Academic Publishers    (1991);-   c) Bundgaard, H., Adv. Drug Deliv. Rev., 8:1-38 (1992);-   d) Nielsen, N. M. et al., J. Pharm. Sci., 77:285 (1988);-   e) Kakeya, N. et al., Chem. Pharm. Bull., 32:692 (1984); and-   g) Rautio, J., ed., Prodrugs and Targeted Delivery (Methods and    Principles in Medicinal Chemistry), Vol. 47, Wiley-VCH (2011).

Compounds containing a carboxy group can form physiologicallyhydrolyzable esters that serve as prodrugs by being hydrolyzed in thebody to yield formula I or formula II compounds per se. Such prodrugsare preferably administered orally since hydrolysis in many instancesoccurs principally under the influence of the digestive enzymes.Parenteral administration may be used where the ester per se is active,or in those instances where hydrolysis occurs in the blood. Examples ofphysiologically hydrolyzable esters of compounds of formula I or formulaII include C₁-C₆alkyl, C₁-C₆alkylbenzyl, 4-methoxybenzyl, indanyl,phthalyl, methoxymethyl, C₁₋₆ alkanoyloxy-C₁₋₆alkyl (e.g.,acetoxymethyl, pivaloyloxymethyl or propionyloxymethyl),C₁-C₆alkoxycarbonyloxy-C₁₋₆alkyl (e.g., methoxycarbonyl-oxymethyl orethoxycarbonyloxymethyl, glycyloxymethyl, phenylglycyloxymethyl,(5-methyl-2-oxo-1,3-dioxolen-4-yl)-methyl), and other well-knownphysiologically hydrolyzable esters used, for example, in the penicillinand cephalosporin arts. Such esters may be prepared by conventionaltechniques known in the art.

Preparation of prodrugs is well known in the art and described in, forexample, King, F. D., ed., Medicinal Chemistry: Principles and Practice,The Royal Society of Chemistry, Cambridge, UK (Second Edition,reproduced, 2006); Testa, B. et al., Hydrolysis in Drug and ProdrugMetabolism. Chemistry, Biochemistry and Enzymology, VCHA and Wiley-VCH,Zurich, Switzerland (2003); Wermuth, C. G., ed., The PracticeofMedicinal Chemistry, Third Edition, Academic Press, San Diego, Calif.(2008).

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include deuteriumand tritium. Isotopes of carbon include ¹³C and ¹⁴C.Isotopically-labeled compounds of the invention can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described herein, using an appropriateisotopically-labeled reagent in place of the non-labeled reagentotherwise employed.

The term “solvate” means a physical association of a compound of thisinvention with one or more solvent molecules, whether organic orinorganic. This physical association includes hydrogen bonding. Incertain instances the solvate will be capable of isolation, for example,when one or more solvent molecules are incorporated in the crystallattice of the crystalline solid. The solvent molecules in the solvatemay be present in a regular arrangement and/or a non-orderedarrangement. The solvate may comprise either a stoichiometric ornonstoichiometric amount of the solvent molecules. “Solvate” encompassesboth solution-phase and isolable solvates. Exemplary solvates include,but are not limited to, hydrates, ethanolates, methanolates, andisopropanolates. Methods of solvation are generally known in the art.

As used herein, the term “patient” refers to organisms to be treated bythe methods of the present invention. Such organisms preferably include,but are not limited to, mammals (e.g., murines, simians, equines,bovines, porcines, canines, felines, and the like), and most preferablyrefers to humans.

As used herein, the term “effective amount” means that amount of a drugor pharmaceutical agent, i.e., a compound of the invention, that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought, for instance, by a researcher or clinician.Furthermore, the term “therapeutically effective amount” means anyamount which, as compared to a corresponding subject who has notreceived such amount, results in improved treatment, healing,prevention, or amelioration of a disease, disorder, or side effect, or adecrease in the rate of advancement of a disease or disorder. Aneffective amount can be administered in one or more administrations,applications or dosages and is not intended to be limited to aparticular formulation or administration route. The term also includeswithin its scope amounts effective to enhance normal physiologicalfunction

As used herein, the term “treating” includes any effect, e.g.,lessening, reducing, modulating, ameliorating or eliminating, thatresults in the improvement of the condition, disease, disorder, and thelike, or ameliorating a symptom thereof.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

Methods of Preparation

The compounds of the present invention may be prepared from startingmaterials which are known in the chemical literature or are commerciallyavailable by methods such as those illustrated in the following Schemesutilizing chemical transformations known to those skilled in the art oforganic chemistry. Solvents, temperatures, pressures, and other reactionconditions may readily be selected by one of ordinary skill in the art.These Schemes are illustrative and are not meant to limit the possibletechniques one skilled in the art may use to manufacture compoundsdisclosed herein. Different methods may be evident to those skilled inthe art. Additionally, the various steps in the synthesis may beperformed in an alternate sequence or order to give the desiredcompound(s). Further, the representation of the reactions in theseSchemes as discrete steps does not preclude their being performed intandem, either by telescoping multiple steps in the same reaction vesselor by performing multiple steps without purifying or characterizing theintermediate(s). In addition, many of the compounds prepared by themethods below can be further modified using conventional chemistry wellknown to those skilled in the art. All documents cited herein areincorporated herein by reference in their entirety.

Reference can also be made to International Application Nos.PCT/US2015/059271, PCT/US2015/059311, and PCT/US2015/059316.

References to many of these chemical transformations employed herein canbe found in Smith, M. B. et al., March's Advanced Organic ChemistryReactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience,N.Y. (2001), or other standard texts on the topic of synthetic organicchemistry. Certain transformations may require that reactive functionalgroups be masked by protecting group(s). A convenient reference whichprovides conditions for introduction, removal, and relativesusceptibility to reaction conditions of these groups is Greene, T. W.et al., Protective Groups in Organic Synthesis, Third Edition,Wiley-Interscience, N.Y. (1999).

Schemes 1-3 depict methods for preparing compounds of formula I. Thesemethods can also be applied to preparation of compounds of theinvention.

Treatment of a phosphonoacetate ester (IV), with a base such as sodiumhydride in a solvent such as THF (Scheme 1) followed by a ketone of thegeneral structure III affords a trisubstituted olefin. Substitutedanalogs of IV (R⁴ is not H) afford tetrasubstitued olefins. This methodand additional methods described below are transformations familiar tothose skilled in the art of organic/medicinal chemistry. Alternativemethods for olefination and the transformations described below areknown and will be selected by one skilled in the art based on theirapplicability to the specific substrate under consideration. Reductionis accomplished by stirring or shaking a solution of the olefin in asuitable solvent under an atmosphere or more of H₂ in the presence of acatalyst, normally palladium on carbon. Hydrolysis of the ketal groupaffords a ketone of the general structure V. Typically, this isaccomplished by heating with an aqueous acid such as HCl in the presenceof a co-solvent such as THF. In addion to the cyclic ethyleneglycol-based ketal shown, other cyclic and acyclic ketal protectinggroups could be used. Ketones are deprotonated with bases such as LiHMDSand react with N-phenyltrifluoromethanesulfonimide or similar reagentsto afford triflates of the general structure VI. These triflatesparticipate in Suzuki couplings (T. Ishiyama, M. Murata, N. Miyaura, J.Org. Chem., 1995, 60, 7508-7510) with boronic acids or esters Ar-B(OR)₂such as IX to afford coupled products. Many variations on this reactionare known, but generally it involves heating the two substrates and acatalyst such as (Ph₃P)₄Pd in a solvent such as DMF with a base such asaq. potassium carbonate. Reduction of the olefin provides intermediate X(where T=a bond). Ketones V can be reduced by NaBH₄ or similar hydridereducing agents to afford alcohols of the general structure VII. Thesealcohols can undergo a Mitsunobu reaction by activation of alcohol VIIwith DIAD, DEAD, or a related azodicarboxylate and a trialkyl ortriarylphosphine and coupling with a heteroaryl alcohol VIII to give X(where T=O). Intermediates X (and later intermediates) may be obtainedas mixtures of cis and trans isomers. Methods for control of thestereochemical outcome of the above reactions are known to thosefamiliar in the art of organic/medicinal chemistry. Additionally,methods for the separation of these isomers are known and described indetail in the synthetic examples. If required, the group R⁴ can beappended by alkylation of intermediate X. Methods for control of theabsolute stereochemistry of the resulting asymmetric center are known tothose familiar with the art, as are chiral separation methods.Saponification of the ester by heating with aq. LiOH or a similar base,generally in the presence of an organic co-solvent such as THF affordscarboxylic acids XI. Acids XI can be rearranged, usually by heating withDPPA and triethylamine (Curtius and related rearrangements), and theintermediate isocyanates react with aq. base to afford primary aminesXII. These can react with electrophiles including, but not limited toacid chlorides and chloroformates of general structure XIII to affordcompounds of the invention.

Piperidine and pyrrolidine esters XIV are known compounds and canundergo S_(N)Ar (T=a bond) reactions with heteroaryl halides of generalstructure XV to afford intermediates XVI(Y═N). These intermediates maybe transformed to compounds of the invention.

Scheme 3 illustrates a method for controlling the absolutestereochemistry of intermediate XXII and materials arising from it.Saponification of esters XVII provides carboxylic acids XVIII. Treatmentof these acids with an acid chloride such as pivaloyl chloride providesa mixed anhydride intermediate. In a separate vessel, an optically pureoxazolidinone of known stereochemistry and general structure XIX isdeprotonated by treatment with a strong base such as n-BuLi. Theseactivated species are combined to form the acyloxazolidinone XX which isdeprotonated by bases such as NaHMDS. Alkylation of the resultingenolate proceeds with predictable control of stereochemistry at thenewly-formed center to provide materials XXI. Removal of the chiralauxiliary to give optically-active carboxylic acids XXII is accomplishedby treatment with a solution of basic hydrogen peroxide. For a review ofthe history and scope scope of this reaction see: D. A. Evans, M. D.Ennis, D. J. Mathre. J. Am. Chem. Soc., 1982, 104 (6), pp 1737-1739.Compounds XXII can be converted to compounds of the invention by methodsshown herein.

EXAMPLES

The following Examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent that the experiments below were performed or thatthey are all of the experiments that may be performed. It is to beunderstood that exemplary descriptions written in the present tense werenot necessarily performed, but rather that the descriptions can beperformed to generate data and the like of a nature described therein.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.), but some experimental errors anddeviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: wt=wildtype; bp=base pair(s);kb=kilobase(s); nt=nucleotides(s); aa=amino acid(s); s or sec=second(s);min=minute(s); h or hr=hour(s); ng=nanogram; μg=microgram; mg=milligram;g=gram; kg=kilogram; dl or dL=deciliter; μl or μL=microliter; ml ormL=milliliter; l or L=liter; μM=micromolar; mM=millimolar; M=molar;kDa=kilodalton; i.m.=intramuscular(ly); i.p.=intraperitoneal(ly); SC orSQ=subcutaneous(ly); QD=daily; BID=twice daily; QW=weekly; QM=monthly;HPLC=high performance liquid chromatography; BW=body weight; U=unit;ns=not statistically significant; PBS=phosphate-buffered saline;IHC=immunohistochemistry; DMEM=Dulbecco's Modification of Eagle'sMedium; EDTA=ethylenediaminetetraacetic acid.

Analytical HPLC/MS was performed using the following methods:

Method A: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%trifluoroacetic acid; Temperature: 50° C.; Gradient: 0-100% B over 3minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection:UV at 220 nm.

Method B: Waters Acquity SDS using the following method: Linear Gradientof 2% to98% solvent B over 1.7 min; UV visualization at 220 nm; Column:BEH C18 2.1 mm×50 mm; 1.7 um particle (Heated to Temp. 50° C.); Flowrate: 0.8 ml/min; Mobile phase A: 100% Water, 0.05% TFA; Mobile phase B:100% Acetonitrile, 0.05% TFA.

Method C: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammoniumacetate; Temperature: 50° C.; Gradient: 0-100% B over 3 minutes, then a0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection: UV at 220 nm.

Example 1(±)-1-(4-Chlorophenyl)-3-(1-(1-(6-fluoroquinolin-4-yl)piperidin-4-yl)ethyl)urea

1A. (±)-tert-Butyl4-(1-(3-(4-chlorophenyl)ureido)ethyl)piperidine-1-carboxylate

To a homogeneous mixture of (±)-tert-butyl4-(1-aminoethyl)piperidine-1-carboxylate (100.0 mg, 0.4 mmol) inanhydrous THF (3 mL), under nitrogen atmosphere, was added DIPEA (0.3mL, 1.7 mmol) followed by 1-chloro-4-isocyanatobenzene (0.1 g, 0.6mmol). The resulting mixture was stirred at ambient temperature for 114hours, before being partitioned between H₂O and EtOAc. The layers wereseparated and the aqueous layer was extracted twice more with EtOAc. Theorganic layers were combined and concentrated in vacuo to afford thetitle compound as a pale yellow solid, which was used in the next stepwithout further purification, based on quantitative yield. MS (ES):m/z=382 [M+H]⁺. t_(R)=1.00 min (Method B).

1B. (±)-1-(4-Chlorophenyl)-3-(1-(piperidin-4-yl)ethyl)urea

To a mixture of (±)-tert-butyl4-(1-(3-(4-chlorophenyl)ureido)ethyl)piperidine-1-carboxylate (1A, 167.0mg, 0.4 mmol) in anhydrous dioxane (5 mL), under nitrogen atmosphere,was added 4M HCl in dioxane (1.1 mL, 4.4 mmol). The resultant mixturewas stirred at ambient temperature for 3.5 hours before beingconcentrated in vacuo to afford the HCl salt of the title compound as awhite solid, which was used without further purification, based onquantitative yield. MS (ES): m/z=282 [M+H]⁺. t_(R)=0.64 min (Method B).1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 7.42 (d, J=8.8 Hz, 2H), 7.22(d, J=8.8 Hz, 2H), 6.89 (d, J=8.8 Hz, 1H), 3.64-3.53 (m, 1H), 3.25-3.19(m, 2H), 2.83-2.71 (m, 2H), 1.83-1.73 (m, 2H), 1.59-1.41 (m, 3H),1.34-1.24 (m, 1H), 1.02 (d, J=6.8 Hz, 3H).

Example 1.(±)-1-(4-Chlorophenyl)-3-(1-(1-(6-fluoroquinolin-4-yl)piperidin-4-yl)ethyl)urea

To a homogeneous mixture of 4-chloro-6-fluoroquinoline (20.0 mg, 0.1mmol) in anhydrous NMP (1 mL), in a sealable vial, was added the HClsalt of (±)-1-(4-chlorophenyl)-3-(1-(piperidin-4-yl)ethyl)urea (1B, 43.4mg, 0.2 mmol) followed by DIPEA (0.07 mL, 0.4 mmol). The vial was sealedand the mixture was stirred at ambient temperature for 2 hours, then at120° C. for 3 hours, before being allowed to cool to room temperature.The reaction mixture was diluted with DMF, filtered through a syringefilter, then purified via preparative HPLC/MS to afford the titlecompound (4.3 mg; 7% yield). MS (ES): m/z=427 [M+H]⁺. t_(R)=1.48 min(Method A). ¹H NMR (500 MHz, DMSO-d₆) δ 8.66 (d, J=4.8 Hz, 1H), 8.50 (s,1H), 8.06-7.95 (m, 1H), 7.65-7.52 (m, 2H), 7.42 (d, J=8.7 Hz, 2H), 7.25(d, J=8.7 Hz, 2H), 7.02 (d, J=4.8 Hz, 1H), 6.20 (d, J=8.6 Hz, 1H),3.77-3.64 (m, 1H), 3.59-3.48 (m, 1H), 2.84-2.67 (m, 2H), 2.58-2.52 (m,1H), 1.92-1.74 (m, 2H), 1.65-1.48 (m, 3H), 1.12 (d, J=6.6 Hz, 3H).

Example 2 (±)-1-(4-chlorophenyl)-3-(6-(6-fluoroquinolin-4-yl)spiro[2.5]octan-1-yl)urea

Intermediate 2A: 1,4-dioxaspiro[4.5]dec-7-en-8-yl trifluoromethanesulfonate

To a stirred solution of 1,4-dioxaspiro[4.5]decan-8-one (300 g, 1920.86mmol, 1.0 eq) and N-phenyltrifluoromethanesulfonimide (823.47 g, 2305.03mmol, 1.2 eq) in MTBE (7.5 L) under nitrogen atmosphere at −78° C. wasadded 2.0 M NaHMDS in THF (1152.2 mL, 2305.03 mmol, 1.2 eq) over 70minutes, and the mixture was stirred for an additional 60 minutes. Thereaction mixture was warmed to room temperature and stirred overnightuntil TLC showed complete consumption of the starting material. Themixture was quenched with aqueous KHSO₄ (100 ml), filtrated to removethe solid and concentrated the filtrate completely. To the residue wasadded 3 L MTBE, then washed with 5% NaOH (1.5 L×3). The organic phasewas concentrated to obtain Intermediate 2A (567 g, light yellow oil,yield 102% yield). TLC Rf: 0.7 (PE/EtOAc=10/1, KMnO4). 1H NMR (400 MHz,CDCl₃): δ (ppm) 5.65 (t, J=4.0 Hz, 1H), 3.98 (d, J=1.5 Hz, 4H), 2.53 (s,2H), 2.40 (s, 2H), 1.90 (t, J=6.6 Hz, 2H).

Intermediate 2B:4,4,5,5-tetramethyl-2-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-1,3,2-dioxaborolane

A mixture of intermediate 2A (600 g,2.08 mol, 1 eq), B₂Pin₂ (687.1 g,2.71 mol, 1.3 eq), KOAc (613 g, 6.24 mol, 3 eq), NaBr (86 g 0.833 mol,0.4 eq) and Pd(dppf)C12 (76 g, 0.1 mol, 0.05 eq) in dioxane (6.5 L) washeated to reflux overnight. Once the reaction was complete, the mixturewas concentrated and purified by via silica gel column chromatography togive Intermediate 2B (369 g, 66% yield). LC-MS Anal. Calc'd forC₁₄H₂₃BO₄ 266.17, found [M+H] 267.1. 1H NMR (400 MHz, CDCl₃) δ 6.46 (s,1H), 3.98 (s, 4H), 2.37-2.35 (m, 4H), 1.74-1.60 (t, 2H), 1.24 (s, 12H).

Intermediate 2C: 6-fluoro-4-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)quinoline

A mixture of Intermediate 2B (368 g, 1.38 mol, 1.3 eq),4-Chloro-6-fluoroquinoline (195 g, 1.07 mol, leq), K₂CO₃ (445 g, 3.22mol,3 eq) and Pd(PPh3)4 (25 g, 22 mmol, 0.02 eq) in dioxane-water (3L,4:1) was heated to reflux overnight. The solution was concentrated andextracted with EtOAc. The crude residue was purified via silica gelcolumn chromatography to give Intermediate 2C (236 g, 77% yield). LC-MSAnal. Calc'd for C₁₇H₁₆FNO₂ 285.12, found [M+H] 286.1. 1H NMR (400 MHz,CDCl3) δ 8.80-8.29 (d, 1H), 8.11-8.07 (q, 1H), 7.63-7.61 (q, 1H),7.47-7.46 (q, 1H), 7.26-7.22 (m, 1H), 5.75-5.74 (m, 1H), 4.08-4.05 (m,4H), 2.63-2.59 (m, 2H),2.59-2.53 (m, 2H), 2.0-1.97 (m, 2H).

Intermediate 2D: 6-fluoro-4-(1,4-dioxaspiro[4.5]decan-8-yl)quinoline

To Intermediate 2C (125 g, 0.44 mol) in IPA (2 L) at 55° C. was added10% Pd/C and the mixture was stirred under an atmosphere of H₂overnight. The mixture was filtered and concentrated to give crudeIntermediate 2D (128 g, 100% yield), which was used directly in the nextstep. LC-MS Anal. Calc'd for C₁₇H₁₈FNO₂ 287.13, found [M+H] 288.2,rt=0.62 min (method B)

Intermediate 2E: 4-(6-fluoroquinolin-4-yl)cyclohexan-1-one

Intermediate 2D (100 g, 0.348 mol) was treated with 4 N HCl (300 mL) inacetone (1200 mL) at 45° C. for overnight. Then the solution wasconcentrated in vacuo. The residue was adjusted pH 9 with 6 N NaOH. themixture was partitioned between ethyl acetate and water. The organiclayer was washed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated to give light yellow solid, which was then purified bysilica gel column chromatography to afford intermediate 2E as whitesolid, (67 g, 55% yield). LC-MS Anal. Calc'd for C₁₅H₄FNO 243.11, found[M+H] 244.0. 1H NMR (400 MHz, CDCl3) δ 8.84 (d, J=4.6 Hz, 1H), 8.16 (dd,J=9.3, 5.7 Hz, 1H), 7.72 (dd, J=10.3, 2.8 Hz, 1H), 7.52 (ddd, J=9.2,7.8, 2.7 Hz, 1H), 7.29 (d, J=4.6 Hz, 1H), 3.69 (ddd, J=12.1, 9.0, 3.3Hz, 1H), 2.77-2.54 (m, 4H), 2.37 (ddd, J=13.4, 5.9, 3.0 Hz, 2H), 2.04(qd, J=12.6, 5.3 Hz, 2H).

Intermediate 2F: ethyl2-(4-(6-fluoroquinolin-4-yl)cyclohexylidene)acetate

Sodium hydride (0.104 g, 4.32 mmol) was added in portions to a solutionof ethyl 2-(diethoxyphosphoryl)acetate (0.968 g, 4.32 mmol) in DMF (4.11ml) at 0° C. After the addition was complete the reaction mixture wasstirred at ambient temperature for 30 minutes before adding a solutionof Intermediate 2E (1.0 g, 4.11 mmol) in DMF (2.0 mL). After 30 minutesat ambient temperature the reaction mixture was poured water andextracted with EtOAc. The organic layer was washed with water, brine,dried over sodium sulfate, filtered and concentrated in vacuo. Theresulting residue was purified by silica gel column chromatography togive Intermediate 2F (1.20 g, 3.83 mmol, 93% yield). LC-MS Anal. Calc'dfor C₁₉H₂₀FNO₂ 313.15, found [M+H] 314.2, RT=0.77 min (method B). ¹H NMR(400 MHz, CHLOROFORM-d) δ: 8.80 (d, J=4.6 Hz, 1H), 8.13 (dd, J=9.3, 5.7Hz, 1H), 7.70 (dd, J=10.5, 2.8 Hz, 1H), 7.48 (ddd, J=9.2, 7.9, 2.8 Hz,1H), 7.25 (d, J=4.6 Hz, 1H), 5.74 (s, 1H), 4.18 (q, J=7.2 Hz, 2H), 3.46(tt, J=12.0, 2.9 Hz, 1H), 2.46-2.55 (m, 2H), 2.12-2.24 (m, 3H),1.63-1.81 (m, 3H), 1.30 (t, J=7.1 Hz, 3H)

Intermediate 2G: ethyl(±)-6-(6-fluoroquinolin-4-yl)spiro[2.5]octane-1-carboxylate

To a solution of sodium hydride (40.2 mg, 1.005 mmol) in THF (1596 μl)at room temperature, trimethylsulfoxonium iodide (242 mg, 1.101 mmol)was added as a solid in small portions. The mixture was stirred for 1hour before a solution of Intermediate 2F (300 mg, 0.957 mmol) in DMSO(0.96 mL) was added. The mixture was stirred at 50° C. for 12 hours. Thereaction was concentrated in vacuo, diluted with 5 mL water andextracted with DCM. Combined organics were concentrated and cruderesidue purified via silica gel column chromatography to giveIntermediate 2G (211 mg, 0.644 mmol, 67.3% yield) (Mixture ofdiastereomers and enantiomers). LC-MS Anal. Calc'd for C₂₀H₂₂FNO₂327.16, found [M+H] 328.3; RT=0.78 min (method B).

Intermediate 2H:(±)-6-(6-fluoroquinolin-4-yl)spiro[2.5]octane-1-carboxylic acid

To a solution of Intermediate 2G (211 mg, 0.644 mmol) in THF (2578 μl),Water (2578 μl), and Methanol (1289 μl) at room temperature, lithiumhydroxide (154 mg, 6.44 mmol) was added and the reaction stirred at roomtemperature. After 1 hour, temperature was increased to 40° C. After 18hours, the reaction was concentrated in vacuo and 1N HCl was added toacidify. Mixture was extracted with EtOAc, dried with sodium sulfate,filtered, and concentrated in vacuo to give crude Intermediate 2H (113mg, 0.377 mmol, 58.6% yield) (mixture of diastereomers and enantiomers).Material contaminated with some unreacted SM from A06BA-068. LC-MS Anal.Calc'd for C₁₈H₁₈FNO₂ 299.13, found [M+H] 300.4; RT=0.64 min (method B).

Example 2:(±)-1-(4-chlorophenyl)-3-(6-(6-fluoroquinolin-4-yl)spiro[2.5]octan-1-yl)urea

Intermediate 2H (30 mg, 0.100 mmol) (and diastereomer) was taken up inDioxane (1002 μO. Diphenylphosphoryl azide (33.1 mg, 0.120 mmol) andDIPEA (19.25 μl, 0.110 mmol) were added and the reaction was stirred atroom temperature for 1 h. The reaction was then heated to 50 C and after1 hour, more DPPA (33 mg) was added. After 3 hours, 4-chloroaniline(19.18 mg, 0.150 mmol) was added and the reaction was heated to refluxfor 1 hour. The reaction was concentrated in vacuo, taken up in DMF andpurified via preparative HPLC to give Example 2 (3.7 mg, 0.008 mmol, 8%yield). LC-MS Anal. Calc'd for C₂₄H₂₃ClFN₃O 423.15, found [M+H] 424.1;RT=2.052 min (method C). ¹H NMR (500 MHz, DMSO-d₆) δ: 8.90 (br. s., 1H),8.64 (s, 1H), 8.15 (d, J=9.2 Hz, 2H), 7.77 (t, J=8.7 Hz, 1H), 7.47 (d,J=8.4 Hz, 2H), 7.28 (d, J=8.8 Hz, 2H), 3.42 (br. s., 1H), 2.91 (br. s.,1H), 1.91-2.09 (m, 4H), 1.80 (d, J=11.5 Hz, 1H), 1.60-1.76 (m, 2H), 1.39(d, J=13.1 Hz, 1H), 1.15 (t, J=7.2 Hz, 1H), 1.03 (d, J=12.6 Hz, 1H),0.75 (t, J=6.2 Hz, 1H), 0.40 (br. s., 1H).

Example 3(±)-1-(4-chlorophenyl)-3-((R)-1-((trans)-4-(quinolin-4-yloxy)cyclohexyl)propyl)urea

Intermediate 3A: ethyl 2-(1,4-dioxaspiro [4.5] decan-8-ylidene)acetate

Triethyl phosphonoacetate (21.79 ml, 109 mmol) was added to a suspensionof sodium hydride (3.84 g, 96 mmol) in THF (64.0 ml) and 0° C. Reactionwas stirred at room temperature for 30 minutes. After 30 minutes, thereaction was recooled to 0° C. and a soution of1,4-dioxaspiro[4.5]decan-8-one (10 g, 64.0 mmol) in 5 mL THF was added.The reaction was then stirred at room temperature for 30 minutes priorto quenching with water. The mixture was extracted with DCM three times.Combined organic extracts were dried with sodium sulfate, filtered, andconcentrated in vacuo. Crude residue was purified via silica gelchromatography to give intermeduate 3A (13.88 g, 61.3 mmol, 96% yield).TLC: product stains as purple spot in anisaldehyde (Rf=0.75 in 1:1Hex/EtOAc). ¹H NMR (400 MHz, CHLOROFORM-d) δ: 5.65 (s, 1H), 4.13 (q,J=7.2 Hz, 2H), 3.92-3.99 (m, 4H), 2.94-3.02 (m, 2H), 2.31-2.40 (m, 2H),1.71-1.79 (m, 4H), 1.26 (t, J=7.2 Hz, 3H)

Intermediate 3B: ethyl 2-(1,4-dioxaspiro[4.5]decan-8-yl)acetate

Intermediate 3A (13.88 g, 61.3 mmol) was taken up in EtOAc (61.3 ml) andwas added to a Parr hydrogenation bottle containing wet 10% palladium oncarbon (1.306 g, 12.27 mmol)(54% w/w Water) under an atmosphere ofnitrogen. The reaction bottle was purged and back-filled with nitrogenthree times, and then with hydrogen. After filling the bottle withhydrogen to 50 psi, the bottle was placed in a Parr shaker and shaken.After 4 hours, the reaction was filtered over pressed celite andconcentrated in vacuo to give Intermediate 3B (13.78 g, 60.4 mmol, 98%yield). LC-MS Anal. Calc'd for C₁₂H₂₀O₄ 228.14, found [M+H] 299.1T_(r)=0.83 min (Method B). ¹H NMR (400 MHz, CHLOROFORM-d) δ: 4.11 (q,J=7.2 Hz, 2H), 3.88-3.95 (m, 4H), 2.21 (d, J=7.0 Hz, 2H), 1.83 (dqd,J=11.0, 7.5, 3.5 Hz, 1H), 1.68-1.78 (m, 4H), 1.50-1.61 (m, 2H),1.27-1.35 (m, 2H), 1.24 (t, J=7.2 Hz, 3H)

Intermediate 3C: (±)-ethyl 2-(1,4-dioxaspiro[4.5]decan-8-yl)butanoate

Diisopropylamine (2.347 ml, 16.63 mmol) taken up in dry THF (15.99 ml)(under N2 atmosphere) and cooled to −78° C. n-BuLi (6.14 ml, 15.35 mmol)(2.5 M in hexanes) was added over ˜5 minutes at −78° C. After stirringfor 45 minutes, reaction was warmed to room temperature for 10 minutesand returned to −78° C. Then, 1,3-dimethyltetrahydropyrimidin-2(1H)-one(1.541 ml, 12.79 mmol) was added followed by a solution of Intermediate3B (2.92 g, 12.79 mmol) in THF (15.99 ml) (dropwise over ˜5 minutes).After 1 hour, iodoethane (1.125 ml, 14.07 mmol) (neat) was addeddropwise over ˜5 minutes. Reaction stirred another 2 hours at −78° C.before slowly warming to room temperature. The reaction was then stirredover night at room temperature. The reaction was quenched by pouringinto 1:1 water/brine and extracting with EtOAc. Combined organics washedwith brine, dried with sodium sulfate, filtered and concentrated invacuo. Crude residue was purified via silica gel column chromatographyto give Intermediate 3C (2.27 g, 8.86 mmol, 69% yield). TLC: productstains as purple spot in anisaldehyde (Rf=0.80 in 1:1 Hex/EtOAc). ¹H NMR(400 MHz, CHLOROFORM-d) δ: 4.14 (q, J=7.5 Hz, 2H), 3.88-3.95 (m, 4H),2.09 (td, J=8.4, 5.6 Hz, 1H), 1.69-1.83 (m, 4H), 1.45-1.64 (m, 6H),1.33-1.42 (m, 1H), 1.25 (t, J=7.1 Hz, 3H), 0.86 (t, J=7.5 Hz, 3H)

Intermediate 3D: (±)-ethyl 2-(4-oxocyclohexyl)butanoate

Intermediate 3C (2.00 g, 7.80 mmol) was taken up in THF (39.0 ml) andHydrochloric Acid, 1M (39.0 ml) was added. Reaction stirred at roomtemperature for 2 hours. The reaction was concentrated in vacuo, dilutedwith water and extracted with EtOAc. The combined organic extracts weredried with sodium sulfate, filtered and concentrated in vacuo. The crudematerial was purified on silica gel column chromatography to giveIntermediate 3D (1.47 g, 6.92 mmol, 89% yield). TLC: product stainsfaintly pink in anisaldehyde (Rf=0.65 in 1:1 Hex/EtOAc). ¹H NMR (400MHz, CHLOROFORM-d) δ: 4.15 (q, J=7.1 Hz, 2H), 2.25-2.42 (m, 4H), 2.18(ddd, J=9.3, 7.8, 5.2 Hz, 1H), 2.10 (ddt, J=13.1, 6.2, 3.3 Hz, 1H),1.90-2.03 (m, 2H), 1.56-1.70 (m, 2H), 1.38-1.56 (m, 2H), 1.25 (t, J=7.2Hz, 3H), 0.89 (t, J=7.4 Hz, 3H)

Intermediate 3E: (±)-ethyl-2-((trans)-4-hydroxycyclohexyl)butanoate

Intermediate 3D (1.47 g, 6.92 mmol) was dissolved in EtOH (13.85 ml) andcooled to 0° C. NaBH₄ (0.314 g, 8.31 mmol) was added and the reactionwas then allowed to stir at 0° C. for 1 hour. The reaction was quenchedwith saturated aqueous NH₄Cl and extracted with EtOAc. Combined organicextracts were dried with sodium sulfate, filtered, and concentrated invacuo. The crude material was purified via silica gel columnchromatography to give Intermediate 3E (1.22 g, 5.69 mmol, 82% yield)along with (138 mg, 0.644 mmol, 9.30% yield) of the cis-isomer. ¹H NMR(400 MHz, CHLOROFORM-d) δ: 4.14 (q, J=7.1 Hz, 2H), 3.53 (t, J=10.5 Hz,1H), 1.92-2.08 (m, 2H), 1.80-1.89 (m, 1H), 1.63-1.70 (m, 1H), 1.52-1.62(m, 4H), 1.37-1.52 (m, 2H), 1.26 (t, J=7.2 Hz, 3H), 0.95-1.17 (m, 2H),0.87 (t, J=7.4 Hz, 3H)

Intermediate 3F: (±)-ethyl2-((trans)-4-(quinolin-4-yloxy)cyclohexyl)butanoate

Intermediate 3E (100 mg, 0.467 mmol) was taken up in DMSO (933 μl) andNaH (22.40 mg, 0.933 mmol) as added slowly, portionwise at roomtempreature over 1 minute. After 1 hour, 4-bromoquinoline (117 mg, 0.560mmol) was added and the reaction was heated to 80° C. for 16 hours. Thereaction was quenched with ammonium chloride and extracted with EtOAc.The combined organic extracts were dried with sodium sulfate, filtered,concentrated in vacuo. The crude residue was purified via silica gelcolumn chromatrography to give Intermediate 3F (89 mg, 0.261 mmol, 55.9%yield). LC-MS Anal. Calc'd for C₂₁H₂₇NO₃ 341.20, found [M+H] 342.3T_(r)=0.84 min (Method B).

Intermediate 3G: (±)-2-((trans)-4-(quinolin-4-yloxy)cyclohexyl)butanoicacid

Intermediate 3F (67 mg, 0.196 mmol) taken up in THF (157 μl), Water (157μl), and MeOH (78 μl). Lithium hydroxide (47.0 mg, 1.962 mmol) added andreaction stirred at 60° C. overnight. The reaction was concentrated invacuo, diluted with water and extracted with EtOAc. The aqueous was thentreated with acetic acid and extracted with EtOAc followed by extractionwith 7:3 chloroform:propanol. The combined organic extracts were driedwith sodium sulfate, filtered, and concentrated in vacuo to giveIntermediate 3G (58 mg, 0.185 mmol, 94% yield). Material used as issubsequently. LC-MS Anal. Calc'd for C₁₉H₂₃NO₃ 313.17, found [M+H] 314.3T_(r)=0.69 min (Method B).

Examples 3:(±)-1-(4-chlorophenyl)-3-((R)-1-((trans)-4-(quinolin-4-yloxy)cyclohexyl)propyl)urea

Intermediate 3G (30 mg, 0.096 mmol) was taken up in Toluene (319 μl) anddiphenyl phosphorazidate (29.0 mg, 0.105 mmol) and TEA (16.01 μl, 0.115mmol) were added. The reaction vessel was sealed and heated to 80° C.After about 2 h, the reaction was cooled to rt and 4-chloroaniline(24.42 mg, 0.191 mmol) was added. The reaction was then heated to 110°C. After 1 hour, the reaction was concentrated in vacuo, taken up inDMF, filtered, and purified via HPLC to give Example 3 (14.2 mg, 0.031mmol, 32% yield). LC-MS Anal. Calc'd for C₂₅H₂₈ClN₃O₂ 437.19, found[M+H] 438.2 T_(r)=0.83 min (Method B). ¹H NMR (500 MHz, DMSO-d₆) δ: 8.71(d, J=5.3 Hz, 1H), 8.47 (s, 1H), 8.14 (d, J=8.2 Hz, 1H), 7.93 (d, J=8.3Hz, 1H), 7.76 (t, J=7.6 Hz, 1H), 7.57 (t, J=7.5 Hz, 1H), 7.38-7.43 (m,2H), 7.25 (d, J=8.8 Hz, 2H), 7.15 (d, J=5.5 Hz, 1H), 6.03 (d, J=9.3 Hz,1H), 4.63 (br. s., 1H), 3.45 (br. s., 1H), 2.22 (br. s., 2H), 1.72-1.88(m, 2H), 1.38-1.63 (m, 4H), 1.17-1.38 (m, 3H), 0.87 (t, J=7.2 Hz, 3H)

Example 41-((R)-1-((1s,4s)-4-(6-Fluoroquinolin-4-yl)cyclohexyl)ethyl)-3-p-tolylurea

Preparation 4A:

To a stirred solution of 1,4-dioxaspiro[4.5]decan-8-one (300 g, 1920.86mmol, 1.0 eq) and phenyltrifluoromethanesulfonimide (823.47 g, 2305.03mmol, 1.2 eq) in MTBE (7.5 L) under N2 at −78° C. was added 2.0 M NaHMDSin THF (1152.2 mL, 2305.03 mmol, 1.2 eq) over 70 minutes, and themixture was stirred for an additional 60 minutes. The reaction mixturewas warmed to room temperature and stirred overnight until TLC showedcomplete consumption of the starting material. The mixture was quenchedwith aqueous KHSO₄ (100 ml), filtrated to remove the solid andconcentrated the filtrate completely. To the residue was added 3 L MTBE,then washed with 5% NaOH (1.5 L×3). The organic phase was concentratedto obtain 567 g crude Preparation 4A (light yellow oil, yield 102%). Thecrude can be used directly in next step without further purification.

Preparation 4A: ¹H NMR (400 MHz,CDCl₃): δ (ppm) 5.65 (t, J=4.0 Hz, 1H),3.98 (d, J=1.5 Hz, 4H), 2.53 (s, 2H), 2.40 (s, 2H), 1.90 (t, J=6.6 Hz,2H)

Preparation 4B:

A mixture of crude Preparation 4A (600 g,2.08 mol, 1 eq), B₂Pin₂ (687.1g, 2.71 mol, 1.3 eq), KOAc (613 g, 6.24 mol, 3 eq), NaBr (86 g 0.833mol, 0.4 eq) and Pd(dppf)Cl₂ (76 g, 0.1 mol, 0.05 eq) in dioxane (6.5 L)was heated to reflux overnight. Once the reaction was complete, themixture was concentrated and purified by FCC (2% 410% 420% EtOAc/PE) togive Preparation 4B (369 g, 66%).

Preparation 4B: LC-MS: 267.1 (M+1)+, ¹H NMR (400 MHz, CDCl₃) δ 6.46 (s,1H), 3.98 (s, 4H), 2.37-2.35 (m, 4H), 1.74-1.60 (t, 2H), 1.24 (s, 12H).

Preparation 4C:

A mixture of Preparation 4B (368 g, 1.38 mol, 1.3 eq),4-Chloro-6-fluoroquinoline (195 g, 1.07 mol, leq), K₂CO₃ (445 g, 3.22mol,3 eq) and Pd(PPh₃)₄ (25 g, 22 mmol, 0.02 eq) in dioxane-water (3L,4:1) was heated to reflux overnight. The solution was then concentratedand extracted with EtOAc. Purification by FCC (38% EtOAc/petroliumether) gave Preparation 4C (236 g, 77%).

Preparation 4C: LC-MS: 286.1 (M+1)+, ¹H NMR (400 MHz, CDCl₃) δ 8.80-8.29(d, 1H), 8.11-8.07 (q, 1H), 7.63-7.61 (q, 1H), 7.47-7.46 (q, 1H),7.26-7.22 (m, 1H), 5.75-5.74 (m, 1H), 4.08-4.05 (m, 4H), 2.63-2.59 (m,2H),2.59-2.53 (m, 2H), 2.0-1.97 (m, 2H).

Preparation 4D:

To Preparation 4C (125 g, 0.44 mol) in IPA (2 L) at 55° C. was added 10%Pd/C and the mixture was stirred under an atmosphere of H₂ overnight.The mixture was filtered and concentrated to give crude Preparation 4D(130 g), which was used directly in the next step. Preparation 4E:

Preparation 4D (100 g, 0.348 mol) was treated with 4 N HCl (300 mL) inacetone (1200 mL) at 45° C. overnight. The mixture was monitored by TLC.Then the solution was then concentrated in vacuo. The residue wasadjusted to pH 9 with 6 N NaOH and the mixture was partitioned betweenethyl acetate and water. The organic layer was washed with brine, driedover anhydrous Na₂SO₄, filtered and concentrated to give light yellowsolid, which was then purified by silica gel column using hexanes andethyl acetate (from 20 percent ethyl acetate to 70% ethyl acetate) toafford Preparation 4E as a white solid, (47 g+20 g mixture, yield>55%).Preparation 4E: LC-MS: 244.0 (M+1)+, ¹H NMR (400 MHz, CDCl₃) δ 8.84 (d,J=4.6 Hz, 1H), 8.16 (dd, J=9.3, 5.7 Hz, 1H), 7.72 (dd, J=10.3, 2.8 Hz,1H), 7.52 (ddd, J=9.2, 7.8, 2.7 Hz, 1H), 7.29 (d, J=4.6 Hz, 1H), 3.69(ddd, J=12.1, 9.0, 3.3 Hz, 1H), 2.77-2.54 (m, 4H), 2.37 (ddd, J=13.4,5.9, 3.0 Hz, 2H), 2.04 (qd, J=12.6, 5.3 Hz, 2H).

Preparation 4F:

Intermediate 4E (57.8 g, 237.8 mmol) was dissolved in EtOH (240 mL) andcooled to 0° C. NaBH4 (9.94 g, 261.6 mmol) was added portionwisemaintaining the temperature within a range of 0-10° C. (exothermicreaction). The resulting suspension was stirred for 20 minutes. An LC/MSof an aliquot of the reaction mixture indicated consumption of ketone(m/z (M+H)+=244). The reaction was quenched at 0° C. by the slowaddition of acetone (58 mL) over 15 minutes (exotherm). The reaction waspoured slowly onto 500 mL of saturated aqueous ammonium chloride and 500g of ice. The resulting aqueous solution was extracted with EtOAc (3×300mL) and the combined organic fractions were washed with saturatedaqueous ammonium chloride (250 mL) and saturated aqueous sodium chloride(250 mL). The organic portion was dried over anhydrous sodium sulfateand concentrated under reduced pressure. Sufficient silica to adsorb theoil was added and diluted with 10% MeOH in CH₂Cl₂. A similar quantity ofsilica was used as a silica plug to purify the material. The silica plugwas washed with 10% MeOH in CH₂Cl₂ until UV-active material no longercould be detected by TLC (7:3 EtOAc/Hexanes, R_(f)=0.4). The filtratewas concentrated then suspended in 500 mL of toluene and concentratedagain. Crude Preparation 4F was isolated as a yellow solid (58.2 g) thatwas used in the subsequent step without further purification.

Preparation 4G:

To Preparation 4F (58.2 g, 237.8 mmol) was added MeCN (125 mL) andpyridine (38.7 mL, 480 mmol) and the reaction mixture was cooled to 5°C. using an ice/water bath. Methanesulfonyl chloride (26.0 mL, 336 mmol)was added dropwise at 5° C. (exothermic reaction), the reaction mixturestirred for 1 hr at 5° C. and then brought up to room temperature andstirred for an additional 16 h during which time a white precipitateformed. The heterogeneous mixture was quenched by the addition ofsaturated aqueous ammonium chloride (200 mL) and extracted with CH₂Cl₂(3×300 mL). The combined organic fractions were dried over anhydroussodium sulfate and concentrated under reduced pressure. Excess pyridinewas removed by azeotroping from toluene (3×300 mL). The crude materialwas recrystallized from H₂O/MeOH as follows: 1 mL/mmol of H₂O was addedand the slurry was heated to 120° C. in an oil bath. MeOH was addeduntil the solids went into solution (˜0.5 L). After cooling whitecrystals were collected by filtration to give Preparation 4G (58.6g, >20:1 dr, 76% over two steps). m/z (M+H)+=324.1. ¹H-NMR (400 MHz;CDCl₃): δ 8.82 (dd, J=4.6, 0.2 Hz, 1H), 8.15-8.11 (m, 1H), 7.64-7.61 (m,1H), 7.52-7.46 (m, 1H), 7.25 (s, 1H), 4.78 (tt, J=10.9, 5.2 Hz, 1H),3.24-3.16 (m, 1H), 3.07 (d, J=1.0 Hz, 3H), 2.42-2.38 (m, 2H), 2.16-2.12(m, 2H), 1.93-1.66 (m, 4H).

Preparation 4H:

Di-tert-butyl malonate (33.5 mL, 150 mmol) was added dropwise to astirred suspension of NaH (6.0 g, 60% suspension in oil, 150 mmol) in1,2-dimethoxyethane (100 mL) under Ar, cooled in a water-ice bath. Afterstirring for 10 min, Preparation 28B (16.2 g, 50 mmol) was added and thereaction was heated at 85° C. for 20 h. After this time, acetic acid(100 mL) was added, the reaction flask was fitted with a distillationhead and the temperature was raised to 130° C. 1,2-dimethoxyethane wasdistilled off under atmospheric pressure until the distillate was acidic(˜100 mL). The distillation head was removed, a reflux condenser wasattached, water (20 mL) was added and the reaction heated at 130° C. for12 h. The reaction was concentrated under reduced pressure and pouredonto 200 g of ice and 100 mL of saturated aqueous NaOAc. Preparation 40Hwas isolated as a white solid by filtration and further dried byreflxing with toluene in a Dean-Stark apparatus (11.0 g, 76%). m/z(M+H)⁺=288.2. ¹H-NMR (400 MHz; DMSO-d₆): δ 12.05 (bs, 1H), 8.79 (d,J=4.5 Hz, 1H), 8.06 (dd, J=9.2, 5.8 Hz, 1H), 7.94 (dd, J=11.0, 2.8 Hz,1H), 7.66-7.61 (m, 1H), 7.50 (d, J=4.6 Hz, 1H), 2.41 (d, J=7.6 Hz, 2H),2.28-2.23 (m, 1H), 1.87-1.78 (m, 2H), 1.73-1.64 (m, 6H).

Preparation 4I:

To a solution of Preparation 4H (1.4 g, 4.8 mmol) in THF (15 mL) wasadded NEt₃ (1.3 mL, 9.6 mmol). The reaction mixture was cooled to 0° C.and trimethylacetyl chloride (0.713 mL, 5.8 mmol) was added dropwise andthe resulting solution stirred for 30 min at 0° C. In a separate flask,(R)-4-phenyloxazolidin-2-one (1.01 g, 6.24 mmol) in THF (45 mL) at 0° C.was treated with 1 M LiHMDS solution in THF (dropwise addition of 6.24mL, 6.24 mmol) and stirred at 0° C. The lithiate was added via cannulato the first flask. The reaction mixture was allowed to warm to rt andwas stirred for 3 hours. LC/MS indicated the complete consumption of thestarting carboxylic acid and formation of the desired imide. Thereaction mixture was poured onto saturated aqueous ammonium chloride (50mL) and the layers were separated. The aqueous layer was extracted withEtOAc (3×50 mL). The combined organic extracts were dried over anhydroussodium sulfate and chromatographed on silica using EtOAc/Hexanes 0 to100% gradient to give Preparation 41 as a white foam in 83% yield. m/z(M+H)⁺=433.3. ¹H-NMR (400 MHz; CDCl₃): δ 8.80 (d, J=4.5 Hz, 1H), 8.11(dd, J=9.1, 5.7 Hz, 1H), 7.63 (dd, J=10.5, 2.5 Hz, 1H), 7.48-7.43 (m,1H), 7.40-7.30 (m, 6H), 5.47-5.44 (m, 1H), 4.71 (t, J=8.9 Hz, 1H),4.31-4.28 (m, 1H), 3.20-3.11 (m, 3H), 2.49-2.46 (m, 1H), 1.82-1.67 (m,6H).

Preparation 4J:

A solution of Preparation 4I (21.6 g, 50 mmol) in anhydrous THF (200 mL)was cooled to −40° C. (using acetonitrile/dry ice bath, someprecipitation occurs) and 2 M NaHMDS solution in THF (30 mL, 60 mmol)was added over 5 min (a 5-8° C. rise in temperature was observed). Theresulting yellow reaction mixture was stirred for 10 min, becamehomogeneous, and MeI (10.6 g, 75 mmol) was added dropwise over 2 min (a10° C. rise in temperature was observed). The reaction mixture wasstirred for 1 h at −40° C. and LC/MS indicated the complete consumptionof the starting material and formation of the desired methyl imide. Thereaction mixture was rapidly diluted with saturated aqueous ammoniumchloride solution (400 mL) and the biphasic mixture was stirred for 15min. ^(i)PrOAc (100 mL) was added, the layers were separated, and theaqueous layer was extracted with^(i)PrOAc (3×50 mL). The combinedorganic extracts were dried over anhydrous magnesium sulfate filtered,and concentrated. The resulting residue was recrystallized by dissolvingin 400 mL hot acetone and adding H₂O until a milky solution formedfollowed to re-dissolving with heating (˜3:1 acetone/H₂O). Preparation4J was obtained as white needles (15.04 g, 2 crops, 68%). m/z(M+H)⁺=447.3. ¹H-NMR (400 MHz; CDCl₃): δ 8.81 (d, J=4.6 Hz, 1H), 8.10(dd, J=9.2, 5.7 Hz, 1H), 7.65 (dd, J=10.6, 2.7 Hz, 1H), 7.47-7.42 (m,1H), 7.41-7.29 (m, 6H), 5.47 (dd, J=8.8, 3.8 Hz, 1H), 4.69 (t, J=8.9 Hz,1H), 4.38-4.30 (m, 1H), 4.26 (dd, J=8.9, 3.9 Hz, 1H), 3.26-3.21 (m, 1H),2.18-2.15 (m, 1H), 1.93-1.64 (m, 8H), 1.09 (d, J=6.9 Hz, 3H).

Preparation 4K:

To a solution of Preparation 4J (82.0 g, 183.6 mmol) in THF (610 mL) at0° C. was added aqueous H₂O₂ (35 wt %, 82 mL) and LiOH (7.04 g, 293.8mmol) in H₂O (189 mL). The resulting reaction mixture was allowed toslowly warm to rt and stirred overnight. The reaction was cooled to 0°C. and saturated aqueous sodium bisulfate solution (250 mL) was added.After stirring for 30 min, the THF was removed under reduced pressure.Acetic acid (34 mL) was added followed by EtOAc (300 mL). The layerswere separated, and the aqueous layer was extracted with EtOAc (3×500mL). The combined organic extracts were dried over anhydrous Na₂SO₄,filtered, and concentrated under reduced pressure. The brown crudereaction mixture was suspended in MeCN (400 mL) and the suspension wasbrought to reflux with vigorous stirring. After cooling to rt, thesolids were collected by filtration washing with additional MeCN.Preparation 4K was obtained as a white solid (45.4 g, 82%). m/z(M+H)⁺=302.2. ¹H-NMR (400 MHz; DMSO-d6): δ 12.10 (s, 1H), 8.79 (d, J=4.5Hz, 1H), 8.07 (dd, J=9.2, 5.9 Hz, 1H), 7.97-7.94 (m, 1H), 7.67-7.62 (m,1H), 7.49 (d, J=4.5 Hz, 1H), 3.41-3.36 (m, 1H), 2.73-2.65 (m, 1H),1.83-1.61 (m, 9H), 1.08 (d, J=6.8 Hz, 3H). Chiral HPLC, >99% ee(ChiralPak IC-3, 3 μM, 4.6×250 mm, 15 min isocratic 70% heptane 30%i-PrOH with 230 nm detection) at a flow rate of 0.75 mL/min the desiredenantiomer had a retention time of 8.6 min with the undesired enantiomereluting at 9.5 min.

Preparation 4L:

Preparation 4K (2 g, 6.64 mmol) was taken up in toluene (22.12 ml) anddiphenyl phosphorazidate (2.009 g, 7.30 mmol) and triethylamine (1.110ml, 7.96 mmol) were added. Vial sealed and heated to 70° C. After 2hours, the reaction was cooled to room temperature and concentratedunder reduced pressure. Crude residue was taken up in 40 mL THF and 40mL of water and lithium hydroxide (1.589 g, 66.4 mmol) was added. Thereaction was stirred at room temperature for 1 hour. The reaction wasacidified with 1N HCl (white precipitate forms) and extracted withEtOAc. The aqueous portion was then basified with 1N NaOH (precipitateforms) and extracted with EtOAc 5 times. Basic extracts wereconcentrated in vacuo to give 4 L (1.68 g, 6.17 mmol, 93% yield). LC-MSAnal. Calc'd for C₁₇H₂₁FN₂ 272.17, found [M+H] 273.1 T_(r)=0.50 min(Method B). ¹H NMR (400 Mhz, chloroform-d) δ: 8.80 (d, J=4.6 Hz, 1H),8.11 (dd, J=9.3, 5.7 Hz, 1H), 7.67 (dd, J=10.6, 2.8 Hz, 1H), 7.46 (ddd,J=9.2, 8.0, 2.8 Hz, 1H), 7.32 (d, J=4.5 Hz, 1H), 3.27-3.37 (m, 1H), 3.13(dq, J=9.3, 6.3 Hz, 1H), 2.01-2.10 (m, 1H), 1.67-1.92 (m, 6H), 1.37-1.55(m, 4H), 1.15 (d, J=6.4 Hz, 3H).

Example41-((R)-1-((1s,4s)-4-(6-Fluoroquinolin-4-yl)cyclohexyl)ethyl)-3-p-tolylurea

To a solution of 4 L((R)-1-((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethanamine, 18 mg,0.066 mmol) in THF (1 mL) was added p-tolylisocyanate (9.15 μl, 0.073mmol) (diluted 10 fold in THF). The reaction mixture was stirred at rtfor 1 h. The reaction mixture was concentrated in vacuo and the residuewas dissolved in MeOH, filtered, and purified via preparative HPLC togive Example 4 (12.6 mg, 0.030 mmol, 46.1% yield). LC-MS Anal. Calc'dfor C₂₅H₂₈FN₃O 402.22, found [M+H] 406.2. T_(r)=1.46 min (Method A). ¹HNMR (500 MHz, DMSO-d₆) δ: 8.82 (d, J=4.5 Hz, 1H), 8.20 (s, 1H), 8.08(dd, 5.8 Hz, 1H), 8.00-7.87 (m, 1H), 7.72-7.61 (m, 1H), 7.47 (d, J=4.5Hz, 1H), 7.24 (d, J=8.2 Hz, 2H), 7.00 (d, J=8.1 Hz, 2H), 5.94 (d, J=8.8Hz, 1H), 4.04 (d, J=6.6 Hz, 1H), 2.19 (s, 3H), 1.89-1.75 (m, 5H),1.74-1.50 (m, 5H), 1.12 (d, J=6.4 Hz, 3H)

Examples 5-8

Examples 5-8 were prepared from Intermediate 4 L following the procedurefor

Example 4 using the corresponding isocynate.

Tr (min) Method B* *unless otherwise Ex. No. Name R noted [M + H]⁺Example 5 1-(4-chlorophenyl)-3-((R)-1- ((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethyl)urea

1.50 426.1 Example 6 1-(4-ethoxyphenyl)-3-((R)-1-((1s,4S)-4-(6-fluoroquinolin-4- yl)cyclohexyl)ethyl)urea

1.45 436.2 Example 7 1-(3-chloro-4-methylphenyl)-3-((R)-1-((1s,4S)-4-(6- fluoroquinolin-4- yl)cyclohexyl)ethyl)urea

1.63 440.1 Example 8 1-(4-fluorophenyl)-3-((R)-1-((1s,4S)-4-(6-fluoroquinolin-4- yl)cyclohexyl)ethyl)urea

1.37 410.1

Example 9 p-Tolyl(R)-1-(1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethylcarbamate

To a solution of 4 L((R)-1-((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethanamine, 18 mg,0.066 mmol) in THF (1 mL) was added p-tolyl chloroformate (10.51 μl,0.073 mmol) (diluted 10 fold in THF). The reaction mixture was stirredat rt for 1 h. The reaction mixture was concentrtaed in vacuo and theresidue was diluted with ethyl acetate and saturated NaHCO₃ solution.The organic layer was concentrtaed in vacuo and the residue wasdissolved in MeOH, filtered, and purified via preparative HPLC to giveExample 9 as a TFA salt (18.7 mg, 0.036 mmol, 54% yield). LC-MS Anal.Calc'd for C₂₅H₂₇FN₂O₂. TFA 406.21, found [M+H] 407.1. T_(r)=1.61 min(Method A). ¹H NMR (500 MHz, DMSO-d₆) δ: 8.89 (d, J=4.7 Hz, 1H), 8.14(dd, 5.6 Hz, 1H), 8.10-8.04 (m, 1H), 7.76 (t, J=8.6 Hz, 1H), 7.63 (d,J=9.1 Hz, 1H), 7.58 (d, J=4.7 Hz, 1H), 7.18-7.10 (m, 2H), 6.94 (d, J=8.2Hz, 2H), 3.93-3.85 (m, 1H), 2.26 (s, 3H), 1.93-1.62 (m, 10H), 1.17 (d,J=6.5 Hz, 3H)

Examples 10-11

Examples 10-11 were prepared from Intermediate 4 L following theprocedure for Example 9 using the corresponding chloroformate.

Tr (min) Method B* *unless otherwise Ex. No. Name R noted [M + H]⁺Example 10 4-fluorophenyl (R)-1-((1s,4S)-4- (6-fluoroquinolin-4-yl)cyclohexyl)ethylcarbamate

1.54 411.3 Example 11 4-methoxyphenyl (R)-1-((1s,4S)-4-(6-fluoroquinolin-4- yl)cyclohexyl)ethylcarbamate

1.49 423.3Materials and Methods

The following general materials and methods were used, where indicated,or may be used in the Examples below:

Standard methods in molecular biology are described in the scientificliterature (see, e.g., Sambrook et al., Molecular Cloning, ThirdEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2001); and Ausubel et al., Current Protocols in Molecular Biology,Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y. (2001), whichdescribes cloning in bacterial cells and DNA mutagenesis (Vol. 1),cloning in mammalian cells and yeast (Vol. 2), glycoconjugates andprotein expression (Vol. 3), and bioinformatics (Vol. 4)).

The scientific literature describes methods for protein purification,including immunoprecipitation, chromatography, electrophoresis,centrifugation, and crystallization, as well as chemical analysis,chemical modification, post-translational modification, production offusion proteins, and glycosylation of proteins (see, e.g., Coligan etal., Current Protocols in Protein Science, Vols. 1-2, John Wiley andSons, Inc., N.Y. (2000)).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GCG® Wisconsin Package (Accelrys, Inc., San Diego, Calif.); andDECYPHER® (TimeLogic Corp., Crystal Bay, Nev.).

The literature is replete with assays and other experimental techniquesthat can serve as a basis for evaluation of the compounds describedherein.

An IDO enzyme assay and cellular production of kynurenine (KYN) isdescribed in Sarkar, S. A. et al., Diabetes, 56:72-79 (2007). Briefly,all chemicals can be purchased from Sigma-Aldrich (St. Louis, Mo.)unless specified otherwise. Groups of 1,000 human islets can be culturedfor 24 h in 1 mL medium with cytokines, recovered by centrifugation for5 min at 800×g and sonicated in 150 μL PBS containing a proteaseinhibitor cocktail (Set 2; Calbiochem, EMD Biosciences, San Diego,Calif.). The sonicate can be centrifuged for 10 min at 10,000×g, and thesupernatant can be assayed in triplicate by incubating a 40 μl samplewith an equal volume of 100 mmol/L potassium phosphate buffer, pH 6.5,containing 40 mmol/L ascorbic acid (neutralized to pH 7.0), 100 μmol/Lmethylene blue, 200 μg/mL catalase, and 400 μmol/1L-Trp for 30 min at37° C. The assay can be terminated by the addition of 16 μL 30% (w/v)trichloroacetic acid (TCA) and further incubated at 60° C. for 15 min tohydrolyze N-formylkynurenine to KYN. The mixture can then be centrifugedat 12,000 rpm for 15 min, and KYN can be quantified by mixing equalvolume of supernatant with 2% (w/v) Ehrlich's reagent in glacial aceticacid in 96-well microtiter plate and reading the absorbance at 480 nmusing L-KYN as standard. Protein in the islet samples can be quantifiedby Bio-Rad Protein assay at 595 nm. For the detection of L-KYN in theislet culture supernatants, proteins can be precipitated with 5% (w/v)TCA and centrifuged at 12,000 rpm for 15 min, and determination of KYNin the supernatant with Ehrlich's reagent can be determined as describedabove. IL-4 (10 μg/mL; 500-2,000 units/mL) and 1-α-methyl Trp (1-MT; 40μmol/L) can be added to the incubation media as indicated. This assaycan also form the basis of a cell-based assay, and may be quantified viaLCMS/MS as an alternative to UV/Vis detection.

Western Blot Analyses.

Groups of 1,000-1,200 islets incubated for 24 h in Miami medium in thepresence of cytokines can be harvested and sonicated in PBS as above,and 50 μg protein samples can be electrophoresed on 10% SDS-PAGE gels.COS7 cells (0.6×10⁶ cells/60 mm3 petri dish) transfected with human-IDOplasmid (3 μg) or empty vector cells can be used as positive andnegative controls, respectively. Proteins can be transferredelectrophoretically onto polyvinylidine fluoride membranes by semidrymethod and blocked for 1 h with 5% (w/v) nonfat dry milk inTris-buffered saline and 0.1% Tween and then incubated overnight withanti-human mouse IDO antibody (1:500; Chemicon, Temecula, Calif.),phospho-STAT_(1α) p91, and STAT_(1α) p91 (1:500; Zymed, San Francisco,Calif.). Immunoreactive proteins can be visualized with ECL PLUS®Western blotting detection reagent (Amersham BioSciences,Buckinghamshire, U.K.) after incubation for 1 h with anti-mousehorseradish peroxidase-conjugated secondary antibody (JacksonImmunolabs, West Grove, Pa.).

Immunohistochemical Detection of IDO.

Islets can be fixed in 4% paraformaldehyde in PBS (Invitrogen) for 1 h,immobilized in molten 10% porcine skin gelatin blocks (37° C.), andembedded in optimal cutting temperature compound. Immunofluorescentstaining on islet tissue can be performed on 7 μm sections that werestained with antibodies raised against pancreatic duodenal homeobox 1(PDX1) and IDO. Antigen retrieval can be performed in a water bath for30 min in a buffer containing 10 mmol/1 Tris and 1 mmol/1 EDTA (pH 9.0)at 97° C. The sections can be blocked for 1 h with 5% normal goat serumin PBS. The tissues can then be reacted with mouse monoclonal anti-humanIDO antibody (1:20; Chemicon) and goat polyclonal anti-human PDX1antibody (1:2,000; which may be requested from Dr. Chris Wright, Schoolof Medicine, Vanderbilt, Tenn.) overnight at room temperature in a humidchamber. Secondary antibodies anti-goat (labeled with Cy3) andanti-mouse (labeled with Cy2) can be purchased from Jackson Immunolabsand can be used at a concentration of 1:200. The nuclei can be stainedwith Hoechst 33258 (Molecular Probes, Eugene, Oreg.). Images can beacquired by Intelligent Imaging System software from an Olympus 1X81inverted motorized microscope equipped with Olympus DSU (spinning diskconfocal) and Hamamatsu ORCA IIER monochromatic CCD camera.

Alternative means for evaluating the IDO inhibitors of the presentinvention are described in WO 2010/0233166 and are summarized hereafter.

Biochemical Assay.

cDNA clones for both human and mouse IDO have been isolated and verifiedby sequencing and are commercially available. In order to prepare IDOfor biochemical studies, C-terminal His-tagged IDO protein can beproduced in E. coli using the IPTG-inducible pET5a vector system andisolated over a nickel column. The yield of the partially purifiedprotein can be verified by gel electrophoresis and the concentrationestimated by comparison to protein standards. To assay IDO enzymaticactivity, a 96-well plate spectrophotometric assay for kynurenineproduction can be run following published procedures (see, e.g.,Littlejohn, T. K. et al., Prot. Exp. Purif, 19:22-29 (2000)). To screenfor IDO inhibitory activity, compounds can be evaluated at a singleconcentration of, for example, 200 μM against 50 ng of IDO enzyme in 100μL reaction volumes with tryptophan added at increasing concentrationsat, for example, 0, 2, 20, and 200 μM. Kynurenine production can bemeasured at 1 hour.

Cell-Based Assay.

COS-1 cells can be transiently transfected with a CMV promoter-drivenplasmid expressing IDO cDNA using Lipofectamine 2000 (Invitrogen) asrecommended by the manufacturer. A companion set of cells can betransiently transfected with TDO-expressing plasmid. Forty-eight hourspost-transfection, the cells can be apportioned into a 96-well format at6×10⁴ cells per well. The following day, the wells can be washed and newmedia (phenol red free) containing 20 μg/mL tryptophan can be addedtogether with inhibitor. The reaction can be stopped at 5 hours and thesupernatant removed and spectrophotometrically-assayed for kynurenine aspreviously described for the enzyme assay. To obtain initialconfirmation of IDO activity, compounds can be evaluated at a singleconcentration of, for example, 100 μM. More extensive dose-escalationprofiles can be collected for select compounds.

Pharmacodynamic and Pharmacokinetic Evaluation.

A pharmacodynamic assay can be based on measuring serum levels of bothkynurenine and tryptophan, and calculating the kynurenine/tryptophanratio provides an estimate of IDO activity that is independent ofbaseline tryptophan levels. Serum tryptophan and kynurenine levels canbe determined by HPLC analysis, and serum compound levels can optionallyalso be determined in the same HPLC run.

Compounds can be initially evaluated by challenging mice with LPS andthen subsequently administering a bolus dose of compound at the timethat the serum kynurenine level plateaus. As the kynurenine pool israpidly turned over with a half-life in serum of less than 10 minutes,pre-existing kynurenine is not expected to unduly mask the impact thatan IDO inhibitor has on kynurenine production. Each experiment caninclude non-LPS-exposed mice (to determine baseline kynurenine levelsagainst which to compare the other mice) and a set of LPS-exposed micedosed with vehicle alone (to provide a positive control for IDOactivation). Each compound can initially be evaluated in mice at asingle high i.p. bolus dose in the range of at least 100 mg/kg. Bloodcan be collected at defined time intervals (for example, 50 μL sample at5, 15, 30 min., 1, 2, 4, 6, 8, and 24 hr. following compoundadministration) for HPLC analysis of kynurenine and tryptophan levels(pharmacodynamic analysis) as well as for the level of compound(pharmacokinetic analysis). From the pharmacokinetic data the peak serumconcentration of compound achieved can be determined as well as theestimated rate of clearance. By comparing the level of compound in serumrelative to the kynurenine/tryptophan ratio at various time points, theeffective IC₅₀ for IDO inhibition in vivo can be roughly estimated.Compounds exhibiting efficacy can be evaluated to determine a maximumdose that achieves 100% IDO inhibition at the peak concentration.

EVALUATION OF BIOLOGICAL ACTIVITY

Exemplary compounds were tested for inhibition of IDO activity.Experimental procedures and results are provided below.

HEK293 cells were transfected with a pCDNA-based mammalian expressionvector harboring human IDO1 cDNA (NM 002164.2) by electroporation. Theywere cultured in medium (DMEM with 10% FBS) containing 1 mg/ml G418 fortwo weeks. Clones of HEK293 cells that stably expressed human IDO1protein were selected and expanded for IDO inhibition assay.

The human IDO1/HEK293 cells were seeded at 10,000 cells per 50 μL perwell with RPMI/phenol red free media contains 10% FBS in a 384-wellblack wall clear bottom tissue culture plate (Matrix Technologies LLC)100 nL of certain concentration of compound was then added to each wellusing ECHO liquid handling systems. The cells were incubated for 20hours in 37° C. incubator with 5% CO₂.

The compound treatments were stopped by adding trichloroacetic acid(Sigma-Aldrich) to a final concentration at 0.2%. The cell plate wasfurther incubated at 50° C. for 30 minute. The equal volume supernatant(20 μL) and 0.2% (w/v) Ehrlich reagent (4-dimethylaminobenzaldehyde,Sigma-Aldrich) in glacial acetic acid were mixed in a new clear bottom384-well plate. This plate was then incubated at room temperature for 30minute. The absorbance at 490 nm was measured on Envision plate reader.

Compound IC₅₀ values were calculated using the counts of 500 nM of areference standard treatment as one hundred percent inhibition, andcounts of no compound but DMSO treatment as zero percent inhibition.

Assessment of inhibitor activity in HeLa cell-based indoleamine2,3-dioxygenase (IDO) assay:

HeLa (ATCC® CCL-2) cells were obtained from the ATCC® and cultured inDulbecco's Modified Eagle Medium supplemented with 4.5 g/L glucose, 4.5g/L L-glutamine and 4.5 g/L sodium pyruvate (#10-013-CV, Corning), 2 mML-alanyl-L-glutamine dipeptide (#35050-061, Gibco), 100U/mL penicillin,100 μg/mL streptomycin (#5V30010, HyClone) and 10% fetal bovine serum(#5H30071.03 HyClone). Cells were maintained in a humidified incubatorat 37° C. in 5% CO₂.

IDO activity was assessed as a function of kynurenine production asfollows: HeLa cells were seeded in a 96-well culture plate at a densityof 5,000 cells/well and allowed to equilibrate overnight. After 24hours, the media was aspirated and replaced with media containing IFNγ(#285-IF/CF, R&D Systems) at a final concentration of 25 ng/mL. A serialdilution of each test compound was added to the cells in a total volumeof 200 μL of culture medium. After a further 48 hour incubation, 170 μLof supernatant was transferred from each well to a fresh 96-well plate.12.1 μL of 6.1N trichloroacetic acid (#T0699, Sigma-Aldrich) was addedto each well and mixed, followed by incubation at 65° C. for 20 minutesto hydrolyze N-formylkynurenine, the product of indoleamine2,3-dioxygenase, to kynurenine. The reaction mixture was thencentrifuged for 10 mins at 500×g to sediment the precipitate. 100 μL ofthe supernatant was transferred from each well to a fresh 96-well plate.100 μl of 2% (w/v) p-dimethylaminobenzaldehyde (#15647-7, Sigma-Aldrich)in acetic acid (#A6283, Sigma-Aldrich) was added to each well mixed andincubated at room temperature for 20 mins. Kynurenine concentrationswere determined by measuring absorbance at 480 nm and calibratingagainst an L-kynurenine (#K8625, Sigma-Aldrich) standard curve using aSPECTRAMAX® M2e microplate reader (Molecular Devices). The percentageactivity at each inhibitor concentration was determined and IC₅₀ valuesassessed using nonlinear regression.

Results of the IDO assays are shown in the table below.

HEK Human IDO-1 Example Bio Activity (μM) No. A < 0.05, B < 0.250, C <2.0 1 C 2 C 3 A 4 A 5 A 6 A 7 A 8 A 9 B 10 B 11 A

What is claimed:
 1. A compound of formula I or formula II

wherein X is CH or N; Q is CH or N; R¹ is H, halo, or C₁-C₆haloalkyl;R^(1A) is H, halo, or C₁-C₆haloalkyl; T is a bond or —O—; Y is CH or N;W is —CR⁵—, wherein R⁵ is H or (C₁-C₆alkyl); n is 0, 1, 2, 3, or 4; V isC₁-C₆alkylene optionally substituted with one, two, or three R⁴substituents independently selected from C₁-C₆alkyl and C₃-C₆cycloalkyl;or R⁴ and R⁵, together with the atoms to which they are attached, form aC₃cycloalkyl; R² is H or C₁-C₆alkyl; Z is —NH—, —N(C₁-C₆alkyl), or —O—;R³ is —OH, C₁-C₆alkyl, C₁-C₆haloalkyl, halo, —OC₁-C₆alkyl,—OC₁-C₆haloalkyl, —CN, aryl, or —Oaryl; R^(3A) is H, —OH, C₁-C₆alkyl,C₁-C₆haloalkyl, halo, —OC₁-C₆alkyl, —OC₁-C₆haloalkyl, —CN, aryl, or—Oaryl; or a pharmaceutically acceptable salt thereof, a stereoisomerthereof, a tautomer thereof, or a solvate thereof.
 2. The compound ofclaim 1 that is a compound of formula I.
 3. The compound of claim 1 thatis a compound of formula II.
 4. The compound of claim 1, wherein R¹ isH, F, or —CF₃.
 5. The compound of claim 1, wherein R^(1A) is H.
 6. Thecompound of claim 1, wherein T is a bond.
 7. The compound of claim 1,wherein Y is CH and W is CH.
 8. The compound of claim 1, wherein Y is Nand W is CH.
 9. The compound of claim 1, wherein n is
 2. 10. Thecompound of claim 1, wherein V is C₁alkylene substituted with oneC₁-C₆alkyl substituent.
 11. The compound of claim 1, wherein Z is —NH—or —N(C₁-C₆alkyl).
 12. The compound of claim 1, wherein Z is —O—. 13.The compound of claim 1, wherein R³ is C₁-C₆alkyl, halo, or—OC₁-C₆alkyl.
 14. The compound of claim 1, wherein R^(3A) is H.
 15. Thecompound of claim 1 that is(±)-1-(4-Chlorophenyl)-3-(1-(1-(6-fluoroquinolin-4-yl)piperidin-4-yl)ethyl)urea;1-(4-chlorophenyl)-3-(6-(6-fluoroquinolin-4-yl)spiro[2.5]octan-1-yl)urea;1-(4-chlorophenyl)-3-((R)-1-((trans)-4-(quinolin-4-yloxy)cyclohexyl)propyl)urea;1-((R)-1-((1s,4s)-4-(6-Fluoroquinolin-4-yl)cyclohexyl)ethyl)-3-p-tolylurea;1-(4-chlorophenyl)-3-((R)-1-((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethyl)urea;1-(4-ethoxyphenyl)-3-((R)-1-((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethyl)urea;1-(3-chloro-4-methylphenyl)-3-((R)-1-((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethyl)urea;1-(4-fluorophenyl)-3-((R)-1-((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethyl)urea;p-Tolyl(R)-1-((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethylcarbamate;4-fluorophenyl(R)-1-((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethylcarbamate; or4-methoxyphenyl(R)-1-((1s,4S)-4-(6-fluoroquinolin-4-yl)cyclohexyl)ethylcarbamate; or apharmaceutically acceptable salt thereof, a stereoisomer thereof, atautomer thereof, or a solvate thereof.
 16. A pharmaceutical compositioncomprising a compound of claim 1 and a pharmaceutically acceptablecarrier.
 17. The pharmaceutical compositions of claim 16, furthercomprising ipilimumab, nivolumab, or pembrolizumab, or a combinationthereof.
 18. A method of treating cancer in a patient in need of suchtreatment comprising administering to the patient a therapeuticallyeffective amount of a compound according to claim 1.